Cytotoxic factor as is associated with multiple sclerosis, its detection and its quantification

ABSTRACT

Gliotoxic factor in the isolated or purified state, characterized in that it possesses toxic activity with respect to human or animal astrocytic cells, having the effect of a cytomorphological disorganization of their network of intermediate filaments and/or a degradation of the proteins of said intermediate filaments and/or cell death, in particular by apoptosis.

FIELD OF THE INVENTION

The present invention relates to a cytotoxic factor associated withmultiple sclerosis, characterized by its cytotoxic activity with respectto glial cells, in particular astrocytes, as well as to thedemonstration of this cytotoxic activity in a biological test ofdetection and of monitoring this disease in biological fluids ofpatients suffering, in particular, from multiple sclerosis.

BACKGROUND

Glial cells (astrocytes, oligodendrocytes, microgliocytes) are theprimary or secondary target of pathological processes in variousdiseases of the nervous system, in particular, in man, inleukoencephalitis, leukodystrophies, some forms of encephalopathy, someneurodegenerative diseases such as amyotrophic lateral sclerosis wherethere is a concomitant astrocytic gliosis with neuronal involvement, andlastly inflammatory diseases such as multiple sclerosis or Schilder'sdisease.

Multiple sclerosis (MS) is a chronic disease of the central nervoussystem in man, developing in a succession of phases of remission andexacerbation or according to a steady progression, and theanatomopathological feature of which consists of the formation ofwell-delimited patches of demyelination in the white matter of the brainand spinal cord (1). At histological level, these patches display, atthe early stage of the lesion process, a degradation of the periaxonalmyelin associated with an involvement of the glial cells responsible forthis myelination, the oligodendrocytes (2). An inflammatory macrophageactivation involving microglial cells (tissue macrophages resident inthe central nervous system) as well as, probably, macrophagesoriginating from infiltrated blood monocytes, is associated with thisdemyelination process and contributes to the destruction of themyelinated layers (3). In the center of the demyelinated patch, arelative depletion of glial cells is to be found, whereas aproliferation of astrocytes, or astrocytic gliosis, develops at theperiphery and can, at a later stage, invade the demyelinated plaque togenerate a fibrous or gliotic plaque, as is found on the site of oldlesions (1). These sclerotic structures are the origin of the name givento the disease, multiple "sclerosis" (4).

Another feature of these plaques is their almost invariable associationwith a vascular element around which they appear to develop (5, 1). Athistological level an adverse change in the blood-brain barrier (BBB)consisting of capillary endothelium is commonly observed in them (6). Ineffect, the vascular endothelium of capillary structures is normallybutt-jointed in the central nervous system, except for the fenestratedcapillaries associated with the choroid plexus, periventricularstructures providing for the production of cerebrospinal fluid (7). Thischange in the BBB, marked by a parting of the endothelial cells and bythe uncontrolled passage of a flow of plasmatic fluid and of cells ofblood origin into the neuroglial parenchyma, may be visualized in"active" plaques by the magnetic resonance imaging (MRI) techniquecombined with the intravenous injection, into the patient underexamination, of a solution of gadolinium. This compound enables acontrasted magnetic resonance signal to be obtained in the plasmaticfluid, and makes it possible to detect the abnormal passage of plasmainto the nervous parenchyma with the edema resulting therefrom. It hasbeen possible to show a correlation between the lesion-inducing activityof the plaques and the presence of this edema at the same level, as wellas between the appearance and resorption of this edema and theregression of clinical exacerbations of the disease (8).

One of the decisive factors in maintaining a butt-jointed structure ofthe cerebral capillary endothelium, and hence in the endothelium of theBBB, consists of the underlying presence of cytoplasmic processes in theastrocytes, known as astrocyte feet (6). Probably, these astrocyte feetinduce the formation or permit the maintenance of leakproof junctionstructures (of the zonula occludens type) which provide for the cohesionof the capillary endothelium barrier which is the material expression ofthe BBB. Now, various pathological models report the adverse change inthe BBB associated with a depletion of astrocyte feet (9, 10).

Moreover, in the lesion process of MS, the adverse change in the BBBcontributes to an amplification of the associated inflammatory response,through the afflux, rendered "free", of lymphoid cells originating fromthe blood circulation (11).

The contribution of the inflammation associated with immune cells isconsiderable in MS and participates in the lesion process, in particularvia lymphocytes and self-reacting antibodies (12, 13). However, contraryto the autoimmune animal model of experimental allergic encephalitis,where the involvement of the BBB and the invasion of the nervousparenchyma by lymphoid cells of the circulating blood initiate theneuroglial lesion process (14), it is observed in MS that early lesionprocesses associated with a local macrophage reaction in the nervousparenchyma seem to precede the invasion of the tissue by lymphoid cellsof the circulating blood (15, 3). It is thus apparent that lymphoidcells, and more especially lymphocytes, are not the prerogative ofrecent plaques (16). Furthermore, the density of the lymphocyticinfiltration is most especially pronounced in the perivascular areas andat the periphery of the active plaques of demyelination (11, 3).

The initial stimulus at the origin of MS is at the heart of the debateabout the etiology of MS (17). Arguments have been put forward, in turn,in favor of a viral (18), bacterial (19, 20), autoimmune (21, 13), toxic(22) or genetic (23, 24) hypothesis. In fact, it appears that acombination of a genetic predisposition to the action of a primarypathogenic agent may lead up to a devastating inflammatory andautoimmune process (25).

Different sequences of events may thus explain the demyelination and thefunctional neurological involvement in MS, without it having beenpossible to date to identify a decisive factor which might initiate andgive a coherent explanation to the multitude of fragmented data whichhave accumulated concerning this disease.

Some molecules of bacterial or viral, or even endogenous retroviral,origin are known to possess so-called superantigenic properties (26,27). Their particular properties of direct stimulation of T lymphocytes,specific to different antigens, by binding to the Vβ region of certain"T" receptors, have suggested the hypothesis that such molecules areintimately associated with the etiopathogenic process of MS (28). Now,one of the characteristic effects of these superantigens on T cells isthe premature induction, under certain conditions, of a programmed celldeath or apoptosis (29). Superantigens also have properties of bindingto HLA class II molecules at the surface of cells presenting the antigen(27). It may thus be recalled that astrocytes possess the capacity toexpress HLA class II antigens at their plasma membrane, and inparticular in response to certain pro-inflammatory cytokines such asgamma interferon or tumor necrosis factor alpha (TNF-alpha) (30).However, no superantigen has yet been demonstrated in multiplesclerosis.

Moreover, many "untimely" apoptotic processes, as opposed to the normalapoptotic processes linked, for example, to the development of thenervous system (31), may take place in the absence of superantigen,either in the context of viral infection (32) or in the context ofstimulation of a cell receptor (33). It is also of interest to note thatan apoptosis may be induced by the in vitro stimulation of a TNF-alphamembrane receptor (31), but such a phenomenon has not been studied inthe nervous system, and a fortiori with astrocytes.

Some cytokines may hence trigger a pathogenic process and be produced,in particular by macrophages, and they have a cytotoxic effect onoligodendrocytes (34). It should be noted that TNF-alpha, as well asinterleukin-1-alpha, interleukin-1-beta, interleukin-2, interleukin-6and gamma interferon, do not in principle have a cytolytic effect onastrocytes, but induce, rather, an astrocyte proliferation (35).However, astrocytes can themselves be subjected to stimulations at theorigin of a secretion of TNF-alpha. This may be observed, for example,in response to lipopolysaccharide bacterial toxins or to calciumionophores (36). Thus, an involvement of the oligodendrocytes, and hencea destruction of the myelin layers, may take place indirectly via theTNF-alpha produced by astrocytes. Any molecule which induces such acytokine production by astrocytes, irrespective of its specific effecton the latter (proliferation, differentiation or cytopathogenic effect)is hence potentially demyelinating. TNF-alpha induces anoligodendrocytic cytotoxic mechanism whose primary effects are marked bya swelling of the myelin structures, suggestive of an ionchannel-mediated action (34). A correlation seems, moreover, to existbetween the production of TNF-alpha in vivo and exacerbations of MS(37). The role of astrocytes in such a production of TNF-alpha inpatients suffering from MS is, however, unknown.

At all events, since astrocytes are capable of producing TNF-alpha andof copresenting a target antigen with HLA class II antigens toimmunocompetent cells, which are themselves recruited by a cytopathicand/or inflammatory process, they are, in fact, at the pivotal point ofthe immunopathological interactions such as may be observed in thedemyelinating lesion process which characterizes MS.

Another type of molecule capable of playing a part in the pathogenicprocess leading to the formation of a demyelination plaque, and then toan astrocytic gliosis, consists of the so-called heat shock proteins(HSP) or stress proteins. These proteins constitute a relativelyconserved phylogenetic family and are to be found both in prokaryotesand in higher vertebrates (38). Their synthesis is inducible ineukaryotic cells by various stresses, in particular infectious orthermal stresses, and their interspecies antigenic likeness hassuggested a possible induction of autoimmunity in man by infectiousbacteria carrying HSP, such as Mycobacterium tuberculosis (39).

Bacterial antigens capable of possessing superantigenic properties or ofbeing heat shock proteins have been implicated in the induction ofexacerbations of MS, without it being clearly established to datewhether they are chance cofactors of pathogenicity or are etiologicalagents, or are alternatively pathogens sharing common propertiesresponsible for an identical pathogenicity (40, 42).

This type of mechanism involves, on the one hand, the presence ofexogenous HSP (for example bacterial HSP) against which theimmunocompetent cells become sensitized, and on the other hand theexpression of endogenous HSP (of the infected body) at the surface ofcells expressing HLA class II antigens. If the cellular HSP share commonepitopes with the exogenous HSP, they may be recognized as "infectious"by sensitized lymphocytes. Their role has also been mentioned in MS(42). The "gliotoxicity" of such molecules may proceed via the immuneresponse and, possibly, via the astrocytic cells or the microgliocytes(other macrophage glial cells capable of presenting a specific antigen),but the distinctive cytopathic effects of some HSP on glial cells areunknown in view of the absence of exhaustive studies in this field.

Some investigations carried out have provided arguments in favor of aviral etiology of MS (in particular 43, 44, 45 and WO-93/20188, thecontent of which is incorporated by way of reference).

Following the abovementioned investigations, the present inventors wereled to look for one or more factors which are effectors of thepathogenic process ending in the typical formation of demyelinationplaques and in an astrocytic gliosis.

Although cultures of multiple sclerosis blood monocytes/macrophagescontributed to supporting a viral hypothesis during the abovementionedinvestigations, other aspects of the role of macrophage cells in thepathogenesis of this disease, which do not necessarily involve a viralagent, must be taken into consideration; in this connection, ahypothesis has hence been put forward and verified, according to whichone or more factor(s) which is/are toxic to macroglial cells(astrocytes, oligodendrocytes) might be produced by the monocytes ofpatients suffering from MS.

Although the molecular factors in question are unknown, a toxic factororiginating either from immune or inflammatory cells or from a viral orbacterial agent, or alternatively induced by these latter in thesurrounding cells, is capable of initiating a process ending in an acuteor chronic inflammatory response.

The double uncertainty about the possible retroviral origin of MS (46)and about the possible contribution of a bacterial agent to itspathogenesis (40, 47) confirms the investigators in their search forfactors which are cytotoxic to macroglial cells in patients sufferingfrom MS. In effect, the glial cells are the ones which constitute themain target of the neuropathological process in MS.

A. N. Davison et al. (48) have studied the activity of oligodendrocytes,which are the cells involved in myelin synthesis, and in particular thefactors capable of participating as a myelin synthesis inhibitor. Theseinvestigations are, however, exclusively limited to oligodendrocytes,and do not enable progress to be made in explaining the pathogenicprocess described above.

SUMMARY OF THE INVENTION

Thus, the subject of the invention is a gliotoxic factor, in theisolated or purified state, which possesses toxic activity with respectto human or animal astrocytic cells, said activity having the effect ofa cytomorphological disorganization of their network of intermediatefilaments and/or a degradation of the proteins of said intermediatefilaments and/or cell death, in particular by apoptosis.

Gliotoxic factor is understood to mean a particular molecule, or afactor represented by a set of molecules capable of being defined,displaying biological activity which can be demonstrated by a cytotoxiceffect on glial cells.

The inventors were able to demonstrate that the toxic activity of theabovementioned factor was associated with at least one globularglycoprotein.

According to the invention, glycoprotein is understood to mean a proteinwith which at least one carbohydrate group is combined by covalentbonding.

In addition, they characterized a gliotoxic factor which proves,according to the invention, after successive treatment on an ionexchange resin and then on a column for separation by exclusion, toconsist preponderantly of a light fraction centered around an apparentmolecular weight of approximately 17 KD, and of a less abundant heavyfraction centered around an apparent molecular weight of approximately21 KD, at least said light fraction being resistant, under nondenaturingconditions, to the hydrolytic action of pronase, trypsin or proteinaseK, and each of the two said fractions displaying a strong affinity forlectins and in particular concanavalin A.

The gliotoxic factor according to the invention has a major use in thediagnosis, but also in the prophylaxis and the therapy of MS.

According to the invention, a gliotoxic factor is capable of beingobtained using the method comprising the following steps:

the starting material is a biological sample taken, for example, from apatient suffering from clinically active multiple sclerosis,

said sample is treated successively on an ion exchange resin and then ona column for separation by exclusion, to obtain a gliotoxic factorconsisting preponderantly of a light fraction centered around anapparent molecular weight of approximately 17 KD, and a less abundantheavy fraction centered around an apparent molecular weight ofapproximately 21 KD, each of said fractions possessing gliotoxicactivity and having, in addition, the resistance and/or affinityproperties mentioned above.

Biological sample according to the invention is understood to mean, inparticular, a sample of the fluid, tissue or tissue fragment, mucosa,organ or organ fragment type, or culture supernatant obtained using oneof the abovementioned samples.

Another subject of the invention is a method for detecting and/ormonitoring the activity and/or predicting a pathology such as multiplesclerosis, consisting in detecting, in a biological sample, the presenceand/or the amount of a gliotoxic factor as is defined by the invention.

Preferably, the biological sample containing the gliotoxic factorundergoes a pretreatment process, characterized in that, in order toremove contaminants liable to produce the spurious and nonspecificcytotoxic activity of the gliotoxic factor of the invention, a treatmentof said sample with at least one of the following treatments isperformed:

said sample is brought into contact with protein A,

said sample is brought into contact with an ion exchange resin,

said sample is brought into contact with a lectin and in particularconcanavalin A.

The present invention also relates to a method for detecting and/orquantifying, in a biological sample, the toxic activity of the gliotoxicfactor described above, consisting in incubating said biological samplein a suitable culture medium containing astrocytes, in particularimmortalized astrocytes, enabling them to be cultured, and in detectingand/or quantifying the dead astrocytes and/or the living astrocytes.

To detect and/or quantify the dead astrocytes and/or the livingastrocytes, different assay techniques may be employed, and especiallythe following:

* a calorimetric assay employing calcein-AM and ethidium homodimer,respectively,

* a calorimetric assay employing methyltetrazolium bromide

* a radioactive assay employing ⁵¹ Cr.

According to the invention, another method for detecting and/orquantifying, in a biological sample, the toxic activity of a gliotoxicfactor comprises a step of incubation of said sample in a suitableculture medium containing astrocytes, in particular immortalizedastrocytes, enabling them to be cultured, and a step of detection and/orquantification of the fragmentation of the DNA of the astrocytes and/orof the cytomorphological disorganization of the network of intermediatefilaments and/or of the degradation of the proteins of said intermediatefilaments.

In a method of the invention for detecting, in a biological sample, thepresence of a gliotoxic factor, the properties of the latter areutilized by carrying out a step of capture of said factor by a lectinand in particular concanavalin A, and/or a detection step based on theaffinity of said factor for a said lectin.

Lastly, the final subjects of the invention are diagnostic and/ortherapeutic and/or prophylactic compositions comprising all or part of agliotoxic factor of the invention, natural or synthetic or obtained bygenetic engineering, and/or a ligand specific to said factor.

More generally, the present invention may be applied to a device forcarrying out a biological test using, for example, an ELISA technique,possessing a support or a substrate capable of binding to the factor ofthe invention.

Throughout the text, examples, tables and attached figures, when thediagnosis of MS is mentioned, it is understood to be a diagnosis ofdefinite MS defined by the criteria of Poser (49).

BRIEF DESCRIPTION OF THE DRAWINGS

A better understanding of the invention will be gained on reading thedetailed description which follows, given with reference to the attachedfigures, wherein:

FIG. 1 illustrates the cytotoxic effect, observed under a phase contrastlight microscope, in cultures of rat embryonic brain explants.

FIGS. 2 and 3 show the cytotoxic effect observed on different cell typespresent in the cultures of embryonic explants, by immunocytologicalanalysis using a fluorescence microscope at a suitable wavelength fordetecting a signal emitted by a secondary antibody used in an indirectimmunofluorescence procedure and labeled with fluorescein.

FIG. 4 illustrates the coincidence, in a few cultures of multiplesclerosis monocytes/macrophages, between the reverse transcriptase andcytotoxic activities. The reverse transcriptase activity is given in dpm(disintegration per minute) as a function of the number of days ofculture. The curve plotted as a broken line represents a culture ofmonocytes/macrophages from a patient having a strongly progressivechronic form of MS, the continuous curve with black diamond symbolsrepresents a culture of monocytes/macrophages from a patient having aremitting form of MS in acute exacerbation, and the continuous curvewith open circles represents a culture of monocytes/macrophages from apatient having a remitting form of MS in a period of remission.Cytotoxic activity is represented by a number of plus signs ranging fromone to four (positive cytotoxic effect ranging from very weak to verystrong) or a minus sign (absence of cytotoxicity) opposite the pointplotted on the curve representing the reverse transcriptase activity ofthe corresponding supernatant.

FIG. 5 illustrates, as a function of the days of culture, the kineticsof the expression of cytotoxic activity from culture supernatantsoriginating from patients suffering from MS and from controls. On thegraph, the three curves in continuous or broken lines show the kineticsof gliotoxicity in the culture supernatants of monocytes/macrophagesfrom three different cases of MS, and the points aligned on the abscissaaxis (black triangle, open diamond and open rectangle) the negativekinetics of culture of monocytes/macrophages from three controlssuffering from other neurological diseases.

FIG. 6 shows a visualization of living and/or dead astrocytic cells withan optical system.

FIG. 7 shows the cytological effects linked to cytotoxicity onastrocytes.

FIG. 8 illustrates the mean number of dead (FIG. 8A) or living (FIG. 8B)astrocytic cells after mixing with a monocyte/macrophage culturesupernatant at different dilutions. The means of the counts of the deadand living cells as a function of the dilution of the gliotoxic sampleare represented by black squares on two separate graphs. The confidenceintervals corresponding to each mean are represented by an upper valueand a lower value separated by two standard deviations.

FIGS. 9A and 9B show, respectively, the one- and then thetwo-dimensional acrylamide gel electrophoresis of a gliotoxic fractionobtained from filters used for the in vivo filtration of cerebrospinalfluid (CSF) of patients suffering from multiple sclerosis. Prior toelectrophoresis, the molecules adsorbed on the filter were eluted andthen passed through an ion exchange resin.

FIGS. 10A and 10B show the elution, in 50 mM Tris-HCl buffer, pH 6.8 ona Superose 12 exclusion column, of a gliotoxic fraction obtained afterpassage through DEAE-Sepharose resin and originating from a mixture ofMS monocyte/macrophage culture supernatants sampled at D₆, D₉, D₁₂ andD₁₆ according to FIG. 10A and sampled at D₆, D₉, D₁₃ and D₁₆ accordingto FIG. 10B; the thick line shows the absorption of the proteincomponents at 280 nm with a coefficient of sensitivity of R=1.2; thebroken line shows the gliotoxic activity measured on successivefractions according to the protocol described later in the description.The apparent molecular weights corresponding to the elution volumes (Ve)of the gliotoxicity peaks appear at the top of these peaks.

According to FIG. 10A, elution is done in the absence of urea, andaccording to FIG. 10B, elution is done in the presence of 8M urea.

FIG. 11 illustrates a one-dimensional SDS-polyacrylamide electrophoresisgel showing, in successive wells, the implementation of a proteinpurification of the gliotoxic factor, starting from a crude MSmonocyte/macrophage culture supernatant and ending, after passagethrough a DEAE and then concanavalin A column, with the isolation ofprotein bands of 17 and 21 KD bearing virtually the whole of theactivity of the sample.

FIGS. 12 and 13 illustrate the elutions, on a Superose 12 column in abuffer containing 50 mM Tris-HCl, pH 6.8 with 8M urea, of fractionspreviously obtained in the eluate in pH 3 glycine buffer from a ConA-Sepharose column. These eluates were incubated in the presence ofproteinase K before passage through Superose 12.

FIG. 12 shows a fraction originating from an MS gliotoxic culturesupernatant or monocytes, and FIG. 13 shows a fraction originating froma nongliotoxic control culture supernatant.

The curve shows, in each case, the absorption at 280 nm of the peptidecomponents with a sensitivity of detection R=0.02.

The level of elution of 17-KD standard protein is indicated by an arrow.

FIG. 14 shows the dose-response effect in a ⁵¹ Cr cytotoxicity testafter 72 h of incubation, the dotted curve corresponding to the valuesobtained for an MS monocyte culture supernatant, and the curve in acontinuous line corresponding to the values obtained for a fractionpurified on Con A originating from the same culture supernatant.

FIG. 15 shows the dose-response effect in a methyltetrazoliumcytotoxicity test after 72 h of incubation, the dotted curvecorresponding to the values obtained, in three measurements, for an MSmonocyte culture supernatant, and the curve in a continuous linecorresponding to the values obtained for a fraction purified on Con Aoriginating from the same culture supernatant.

EXAMPLES

The first investigations employed, as biological fluids to be analyzed,the in vitro culture supernatants of monocytes/macrophages from patientssuffering from MS or from healthy controls or those suffering from otherneurological diseases and, as substrate for detection of a cytotoxiceffect, in vitro cultures of explants of rat embryonic cerebral cortex.

Example 1

Culturing of blood monocytes/macrophages

The culture medium comprises RPMI1640 (Boehringer),penicillin-streptomycin (bioMerieux), L-glutamine (bioMerieux), sodiumpyruvate (Boehringer), 100× nonessential amino acids (Boehringer) and ABhuman serum taken from healthy donors seronegative for all virusestransmissible by known blood derivatives.

Lymphoid cells are cultured in 75-cm² Primaria culture flasks (Falcon)after being separated from plasma and the other formed blood elements bycentrifugation on a Ficoll (Lymphoprep®, Flow) gradient. To obtain theselymphoid cells, 50 ml of blood are drawn by venous puncture into aheparinized sterile tube (heparin lithium). The blood and the heparinare mixed well as soon as the blood is drawn. Alternatively, the bloodmay be drawn into tubes containing EDTA. It is then important totransport immediately the tubes maintained at +4° C. to the laboratory,where they will be handled under a "biohazard" laminar flow culture hoodunder sterile conditions. For each sample drawn, an "RPMI" medium isprepared which may advantageously comprise 100-150 ml of RPMI 1640medium, a mixture of penicillin and streptomycin, 4% of L-glutamine, 1%of sodium pyruvate, 1% of Boehringer (100×) nonessential amino acids, aswell as 3 sterile 50-ml conical-bottomed tubes (Falcon) containing 10 mlof the "RPMI" medium described above and 4 sterile 50-ml tubes with 20ml of Ficoll at the bottom. The heparinized tubes are opened in order topipette the blood, deposit it in the tubes containing medium and mix itgently with the medium described above. 5 ml of "RPMI" medium are takenand the wall of the heparinized tubes is rinsed. It is necessary toaccompany this rinse by a gentle scraping using the end of the plasticpipette in order to detach cells which have possibly adhered to thewalls of the tube, and to deposit it sic! in the tubes containing theblood diluted in the "RPMI" medium, mixing the contents gently bysuccessive aspirations/expulsions. These operations should be repeateduntil the heparinized tubes are clean. The blood diluted in the "RPMI"medium should then be deposited very gently (without disturbance) on thesurface of the Ficoll in the 50-ml tubes, and "RPMI" medium should thenbe used to rinse the remaining diluted blood and recover it as above soas to deposit it with great care on the surface of the tubes with theFicoll. Thereafter, and without shaking the Ficoll, the tubes should beplaced in centrifuge buckets, water-filled tubes should be used forbalancing and centrifugation should be carried out at +15° C. for 20minutes at 1800 rpm, with a slow deceleration mode. Aftercentrifugation, the tubes are recovered, a pipette is inserted gentlyinto them to the depth of the upper "Ficoll/plasma" interface, and thewhitish layer located above the Ficoll is aspirated gently whiledescribing concentric circles from the walls, and then describing"zig-zags" from one side to the other of the Ficoll surface. Theaspirated medium is placed in 50-ml tubes, diluted in at least 3 timesthe volume of RPMI medium and mixed gently by inversion of the tubeswhich have been stoppered under sterile conditions. The tubes are thencentrifuged at +15° C. for 10 minutes at 1800 rpm, with a slowdeceleration mode. After centrifugation, the supernatant in these tubesis discarded by pouring slowly but evenly, taking care that the whitishcell pellet does not become detached. The pellet is resuspended in 10 mlof "RPMI" medium by successive aspirations/expulsions, and thesuspension is centrifuged at +15° C. for 10 minutes at 1800 rpm, with aslow deceleration mode. For each sample or for every 50 ml of blooddrawn, two small 75-cm² electropositive plastic culture flasks (Falcon"PRIMARIA"), and 10 ml of "RPMI" medium to which 15% of "AB" human serum(HS) described above has been added, are prepared. After centrifugation,the supernatant is removed as above, the pellet is resuspended gently in5 ml of "RPMI" medium with 15% of HS and the resuspended cells aredistributed in the flasks, which are placed flat and barely raised. Thesuspension is immediately distributed by moving each flask in the flatposition. The centrifugation tubes are rinsed with 5 ml of "RPMI" mediumcontaining 15% HS, and the suspension is added and distributed in thetwo flasks as above. Advantageously, all the media used for these stepsare at 37° C. (warmed on a water bath). When the flasks have beenclosed, they are kept flat in a humid incubator at 37° C. with 5% of CO₂until the following morning. The following morning, all the supernatantwith the cells in suspension should be thoroughly aspirated, and flasksshould be rinsed twice with 4 ml of RPMI alone, allowing 5 minutes eachtime for "soaking" and standing the flask up slowly before aspiratingall the remaining medium in order to remove nonadherent cells. Theflasks are then filled with 5 ml of RPMI medium containing 15% of HS andare replaced in the incubator, and care is taken not to disturb them for4 h. From this step onwards, the flasks placed upright should always befilled by directing the jet on to the upper wall in order not to detachthe cells which are in the process of adhering, and then, subsequently,affected by a possible cytopathogenic effect. The cell suspensions thuscollected 24 h after setting up the culture are centrifuged at +15° C.for 10 minutes at 1800 rpm, with a slow deceleration mode. Whereappropriate, the cell pellet may be taken up in fetal calf serum with10% of DMSO (dimethyl sulfoxide) and frozen at -80° C. or in liquidnitrogen according to a procedure for maintaining viable cells. Thecorresponding supernatant is then centrifuged at 3000 rpm for 30 min. inorder to remove cell debris, and the clarified supernatant is aliquoted,listed as a sample at 24 h of culture, that is to say D1, and thenstored in the freezer at -80° C. After 48 h in the incubator, the flasksare taken out, and the supernatant is aspirated with great care and, asabove, centrifuged at 3000 rpm for 30 minutes in order to remove celldebris. The clarified supernatant is aliquoted, listed as a sample at 3days of culture, that is to say D3, and then stored in the freezer at-80° C. The flasks are immediately filled with 5 ml of RPMI mediumcontaining 5% HS and replaced in the incubator. From this point onwards,the culture medium now contains only 5% of HS, and this proportion willbe used for all the renewals of medium. The media in the flasks are thenwithdrawn, stored in aliquots of medium cleared of cell debris, at -80°C. as above, and replaced by "RPMI" medium containing 5% of HS everythree or four days until there no longer persists any adherent cell inthe flask causing refraction on microscopic observation.

Example 2

Cytotoxicity test on cultures of explants of rat embryonic cerebralcortex and explants of rat embryonic spinal cord.

Cultures of explants of rat embryonic cerebral cortex are obtained frombrains of rat embryos taken from pregnant rats at the fourteenth day ofembryonic life. After dissection, the brains are rinsed three times in"Dulbecco-Phosphate Buffer Saline lacuna! (D-PBS: KH₂ PO₄ 0.2 g/l, Na₂HPO₄.7H₂ O 1.15 g/l, NaCl 8 g/l, KCl 0.2 g/l) buffer, and then in F12medium (Boehringer). After the meninges have been removed with greatcare and the cortex isolated, the latter is broken up mechanically usingscissors in F12 medium. Concomitantly, spinal cord explants wereprepared and cultured according to the same principle. The volume isthen adjusted to 30 ml of F12 medium supplemented with 7.5% of fetalcalf serum and 7.5% of horse serum. After 10 minutes of settling, thesupernatant is centrifuged for 5 minutes at 4000 rpm. The pelletobtained is suspended in 10 ml of complete medium. The cells are platedout at a density of 10⁶ cells per sterile dish 35 mm in diameter(Falcon) and maintained at 37° C. under 7.5% of CO₂ and at 95% humidity.Alternatively, slides with culture cavities of the Labtek® type may beused, in particular for a subsequent immunohistochemical study, afterfixing the culture at the requisite time. The culture medium is changedevery three days. Usually after 3 to 7 days, when a good differentiationof the cortical neurons is obtained with a well-balanced organization ofthe underlying lawn of glial and leptomeningeal cells (a little piamater remains associated with the sample of cerebral tissue used), thecultures are used for cytotoxicity tests with respect to cells of thecentral nervous system.

The test samples are heated for 30 min at 56° C. in order to inactivatecomplement proteins and a possible enveloped virus, and then centrifugedfor 10 minutes at 1500 rpm. Where appropriate, the recovered supernatantis dialyzed at 40° C. in twice 20 volumes of D-PBS buffer, a first timefor 2 h and a second time overnight. The test sample is aliquoted andstored at -20° C. An aliquot is thawed for the cytotoxicity tests andmixed according to the desired dilution with the culture medium of thetarget cells, and the mixture is replaced in the incubator. In thisinstance, the target cells consist of all of the cells present in theculture of the explant of cerebral tissue.

A considerable cytotoxic effect was observed under these conditions withsupernatants diluted to 1/4 in the culture medium of the explantsdescribed above, and sampled between the third and the tenth day ofculture of blood monocytes from patients suffering from MS in acuteexacerbation. This effect was observable under the light microscope usedfor examination of the cells in culture, from the sixth hour followingthe introduction of the diluted sample into the culture medium. Theeffect was first significant in the glial cells, identified by theiraltogether typical morphology during a regular observation of theculture. A swelling of the oligodendrocytes, which ended up by roundingoff and detaching themselves from the culture support, was thusobserved, together with a vacuolization of the astrocytes accompanied bya considerable regression of their cytoplasmic processes. At this stage,the neurons present in the culture were relatively well preserved and donot display significant adverse change. Subsequently, afterapproximately 24 hours under these conditions, the glial lawn beingcompletely destroyed, the neurons ended up by degenerating and detachedthemselves, in their turn, from the culture. After 48 hours under theseconditions, almost no more viable cells remained in the culture wellsbrought into contact with the supernatants ranging from D3 to D12,whereas the effect of the supernatants of the same cultures of monocytesfrom MS in acute exacerbation, sampled on D1, D18 or D21, displayed alesser or even nonexistent effect sic!. The culture supernatants ofblood monocytes/macrophages from a few controls not suffering from MSdid not, in parallel, induce any cytotoxic effect under the sameconditions.

Example 3

Cytotoxicity test on cultures of explants of rat embryonic brain.

Culture supernatants of monocytes/macrophages from MS in acuteexacerbation or from a healthy control were diluted to 1/4 in theculture medium of the brain explants of rat embryos which had reached anadvanced stage of differentiation of the neurons, and then incubated at37° C. as described above. An example of the cytotoxic effect describedabove observed under a light microscope in the cultures of embryonicexplants is shown in FIG. 1. Photograph A shows a brain cell cultureincubated for 48 h with a sample taken at D6 originating from a healthycontrol. Photographs B and C show two brain cell cultures incubated for24 h with two samples taken, respectively, at D18 and at D6, originatingfrom the same culture of monocytes/macrophages from MS in acuteexacerbation. Photograph D shows a brain cell culture incubated for 48 hwith a sample taken at D6 originating from the same culture ofmonocytes/macrophages from MS in acute exacerbation as for photograph C.

Moreover, an example of the effect observed on the different cell typespresent in the cultures of embryonic explants, by immunocytologicalanalysis using a fluorescence microscope, is shown in attached FIGS. 2and 3. In this example, the supernatants of cultures ofmonocytes/macrophages from MS in acute exacerbation or from a healthycontrol were diluted to 1/4 in the culture medium of explants of brainor of spinal cord of rat embryos, cultured on Labtek® slides asdescribed above. The slides were fixed at the requisite time, after twowashes in PBS, by incubation for 10 minutes in a mixture of equalvolumes of acetone and methanol at -20° C., and then incubated overnightat +4° C. with the appropriate dilution of a first antibody specific forthe cell type to be labeled, that is to say an anti-neurofilamentantibody (Boehringer anti-NF antibody) for neurons, an anti-glialfibrillar acid protein antibody (Boehringer anti-GFAP antibody) forastrocytes and an anti-myelin basic protein antibody (Boehringeranti-MBP antibody) for oligodendrocytes. After two 10-minute washes inPBS followed by a 5-minute wash in distilled water, the slides wereincubated for one hour at room temperature in an appropriate dilution ofa second antibody, specific for the immunoglobulins of the species usedfor producing the first antibody, and coupled to a fluorochrome. Afterwashing the slides as above, the latter were mounted for examinationunder a fluorescence microscope with the appropriate wavelength. In FIG.2, photographs A and B show, magnified 40 times, the labeling with ananti-neurofilament antibody of a spinal cord explant incubated with aculture supernatant of monocyte/macrophage from MS in acuteexacerbation, sampled at D9, for 12 and 48 hours, respectively. In FIG.2, photographs C and D show, magnified 40 times, a spinal cord explantincubated for 24 hours with a culture supernatant of monocyte/macrophagefrom MS in acute exacerbation, sampled at D9, and labeled with ananti-neurofilament antibody and an anti-GFAP antibody, respectively. InFIG. 3, photographs A and B show, magnified 40 times, a spinal cordexplant incubated for 24 hours with a culture supernatant ofmonocyte/macrophage from MS in acute exacerbation, sampled at D6, andlabeled with an anti-MBP antibody and an anti-GFAP antibody,respectively.

The earliest and largest cytotoxic effect in these primary cultures ofcells of the central nervous system hence manifestly relates to themacroglial cells, namely astrocytes and oligodendrocytes.

Example 4

Cytotoxic and reverse transcriptase activities.

The monocyte/macrophage culture protocol having first been developed forstudying a retroviral expression in MS (45), the reverse transcriptaseactivity was tested in a few supernatants according to the conditionspreviously determined for the study which formed the subject of theinvestigations of Perron H. (44), in parallel with their cytotoxicactivity towards brain cells cultured as explants as described above. Ina few MS monocyte/macrophage culture supernatants sampled between D1 andD22, a relative coincidence is observed between the maximum gliotoxicactivity occurring in the culture supernatants and the peak of reversetranscriptase activity. However, since neither heating for 30 minutes at56° C. nor two cycles of freezing/thawing nor removal of the pelletsedimented by ultracentri-fugation at 100,000 g for 2 hours impaired thecytotoxic effect of the supernatants analyzed, a direct effect due toinfection of the explanted cells by a retrovirus present in the MSmonocyte/macrophage culture supernatants is implausible.

The relative coincidence, in a few MS monocyte/macrophage cultures,between the reverse transcriptase and cytotoxic activities is shown inFIG. 4. MS monocyte/macrophage culture supernatants were diluted to 1/4in the culture medium of brain explants, and incubated for 24 hoursaccording to the protocol described above before estimating thecytotoxic effect by microscopic quantification of cell depletion. Theseresults show a relative coincidence between reverse transcriptaseactivity and the observed cytotoxic activity.

Example 5

Cytotoxicity/gliotoxicity tests

In view of the results described above, a standardized system forstudying and quantifying the observed cytotoxic effect was sought. Afterseveral evaluations, it was found that continuous cultures ofimmortalized astrocytes (50) constitute an appropriate material fordetecting and quantifying, on pure and homogeneous glial cells, acytotoxic activity such as is described above.

Thus, the continuous astrocyte line is maintained in DMEM-F12 (1:1)medium supplemented with 10% of FCS (fetal calf serum) in an incubatorwith 7.5% of CO₂, at 37° C. and at 95% humidity. The cells are culturedin culture plates or flasks, previously coated with poly-L-lysine at aconcentration of 5 μg/ml in PBS. The passage density is, in general,2×10³ cells/cm². Under these conditions, the cells are cultured for twodays, or until a homogeneous monolayer cell lawn is obtained, beforebeing used for the cytotoxicity tests. They may advantageously becultured on 24-well culture plates, thereby making it possible to workin series on numerous tests to be performed. For the subsequent studieson slides, they may be cultured on Labtek® type cavity culture slides.Generally speaking, samples originating from biological fluids to betested are heated at 56° C. for 30 min. in order to inactivatecomplement proteins and a possible enveloped virus, and then centrifugedfor 10 minutes at 1500 rpm, and the supernatant recovered.

Each sample is then diluted in the requisite proportion in the DMEM-F12(1:1) culture medium containing 10% of FCS of the abovementionedastrocytes, and the medium homogenized by pipetting or gentle agitationis deposited in the culture flask or well as replacement for themaintenance medium. The cells are then replaced in the incubator underthe conditions described above. The cytotoxic effect with respect toglial cells, represented by astrocytes, is thus assessed in terms ofgliotoxic activity.

The gliotoxic effect of the dilutions of the test samples was measuredby three different techniques which proved to be concordant.

The first technique, known as "L/D test" (living cells/dead cells test),is a rapid, semiquantitative calorimetric assay permitting simultaneousdetermination of living and dead cells. Living cells are distinguishedby the presence of an intracellular esterase activity. The esteraseactivity is detected by a green fluorescence generated by enzymatichydrolysis of a substrate, calcein-AM. Dead cells are distinguished by alabeling in the nucleic acids with ethidium homodimer, and only thenuclei of cells whose nuclear membranes are damaged fluoresce red. After72 h of incubation at 37° C., the cells are rinsed in D-PBS buffer, andincubated for 15 minutes at room temperature and protected from light inthe presence of 200 μl of PBS solution containing 2 μM calcein-AM and 4μM of ethidium homodimer. Observation of the cultures with afluorescence microscope is then performed shortly thereafter. Green(living) and red (dead) cells are counted simultaneously on the samefield of observation. Several fields are observed (at least three), andthe mean of the counts for the dead cells and for the living cells istaken as the result. The gliotoxic (or, more generally, cytotoxic)activity is expressed according to the formula: % cytotoxicity=(meannumber of dead cells/mean number of living cells)×100.

The second technique consists of a calorimetric assay of living cellsusing methyltetrazolium. Methyl-tetrazolium bromide (MTT) is a salt usedfor a quantitative colorimetric assay (51). MTT is a substrate formitochondrial dehydrogenases which, after reduction in metabolicallyactive cells, gives a colored product, formazan (violet). It thusenables living cells to be labeled. Cells (2×10³ cells/cm²) are exposedfor 72 hours to different dilutions of purified fractions (3 wells ordishes per dilution to be tested). They are then rinsed in D-PBS bufferand thereafter incubated for 3 h at 37° C. in 3 ml of MTT solution at aconcentration of 0.5 mg/ml in DMEM-F12 (1:1). The supernatant is removedand acid isopropanol (4×10⁻² M HCl) is applied to the cells in order tosolubilize the formazan crystals. The resulting lysate is centrifugedfor 2 min/6500 rpm in order to remove cell debris. An optical densityreading is then performed at 570-630 nm.

The third technique employs a radioactive assay with ⁵¹ Cr which permitsthe quantification of dead cells. ⁵¹ Cr is a radioactive element capableof entering living cells and which is released into the extracellularmedium only when the cells die. The measurement of incorporatedradioactivity, after washing the cells brought into contact with amedium containing ⁵¹ Cr, as well as that released by the cells into themedium, will permit a quantification of the living and dead cells.Astrocytes (2×10³ cells/cm²) are incubated for 2 h at 37° C. in DMEM-F12(1:1) medium containing 20 μCi of ⁵¹ Cr. The cells are then rinsed 3times in DMEM-F12 medium, exposed to different dilutions of purifiedfractions (3 wells or dishes per dilution to be tested) and incubatedfor 72 h at 37° C. The supernatant is recovered and the cells lysed with1M NaOH solution. Counting, in counts per minute (cpm), of theradioactivity (gamma counter) present in the supernatant and in thelysate is then performed, and a percentage cytotoxicity is determined,calculated in the following manner: % cytotoxicity= (cpmsupernatant--natural cpm)/(total cpm--natural cpm)! ×100. The naturalradioactivity in cpm corresponds to the back-ground measured by thecounter on a sample originating from a culture incubated with the same,nongliotoxic fluid (CSF, serum or monocyte/macrophage culturesupernatant) and with ⁵¹ Cr, under the same conditions as the testsample.

For reproducibility and reliability studies, several wells wereincubated with the same dilution of the same gliotoxic sample, and thestandard deviation obtained was taken into account for the significanceof the results of the subsequent series of analyses. This type ofverification may be performed with each change of batch of astrocyticcells (thawing of a new ampoule) or with each change of batch of culturemedium (batch of FCS most particularly). In the experiments mentionedhere, a percentage of dead cells of greater than 5% proves to beindicative of a cytotoxic effect which is absent under normal cultureconditions.

Example 6

Kinetics of cytotoxic activity

MS monocyte/macrophage culture supernatants which were cytotoxic onprimary cultures of embryonic brain explants, as well as noncytotoxiccontrol supernatants, were tested on cultures of the abovementionedastrocyte line. The presence of gliotoxic activity detected by theastrocytes and visualized by the "L/D test" was to be found in thesupernatants which were previously cytotoxic for the primary culturesand not in those which had not had this property. It was thus possibleto study the kinetics, during the maintenance of monocytes/macrophagesin culture, of the expression of gliotoxic activity in the supernatantsof cultures originating from patients suffering from MS and fromcontrols. It was thus possible to verify that this activity was notdetected at any time in the cultures originating from healthy controls,and also that its intensity fluctuated with time in the "positive"cultures originating from patients suffering from MS. In effect, severalpeaks of activity one sic! been able to be observed in the few casesstudied. These peaks lay, in general, between the 6th and the 9th andthen between the 15th and the 18th day of culture (D6 to D9 and D15 toD18), thus indicating an active synthesis in the culture, since thesupernatant is completely withdrawn therefrom twice weekly at eachchange of culture medium.

An example of such an observation, carried out by means of the use ofthe biological test developed, is shown in FIG. 5. In this example, thesupernatants of MS monocyte/macrophage cultures were treated and used asdescribed above for the cytotoxicity/gliotoxicity tests on theabovementioned astrocyte line with a dilution to 1/10 in the culturemedium, and incubated for 72 hours according to the protocol describedabove with measurement of the gliotoxic effect by the "L/D test" method.

Example 7

Demonstration of a gliotoxic activity in biological fluids of patientssuffering from MS.

Subsequently, this biological test was used to test for possiblegliotoxic activity in the cerebrospinal fluid (CSF) and the serum. Thus,the inventors were able to demonstrate significant gliotoxic activity inthe CSF of patients suffering from MS, and not in CSF of controlssuffering, for example, from normal pressure hydrocephalus (NPH). Thesame finding was made with the corresponding sera, albeit with arelatively lower gliotoxic activity than in the CSF, thereby supportingthe view of an intrathecal production of the factor bearing thisactivity.

An example of the use of the "L/D test" for visualizing the detection byastrocyte targets of a gliotoxic factor in the samples mixed with theculture medium is shown in attached FIG. 6. In this example, a CSF fromremitting MS in exacerbation was diluted to 1/10 in the culture medium,incubated for 72 hours and examined with a fluorescence microscope, theentire procedure being according to the protocol described above for the"L/D test". Photograph A shows a visualization of the living cells withan appropriate optical system for the light emissions from fluorescein(the bar represents 25 micronmeters sic!). Photograph B shows avisualization of the dead cells with an appropriate optical system; thebar represents 25 micronmeters sic!. Photograph C shows a simultaneousvisualization of the living and dead cells with an appropriate opticalsystem for the light emissions from the two fluorophores (485-500 nm);the bar represents 25 micronmeters sic!.

Once this quantification test had been developed, the inventors analyzedfilters prepared by the firm Pall and used in therapeutic trials onpatients suffering from MS (52). These trials consist in filtering thecerebrospinal fluid through filters having, in particular, an affinityfor glycoproteins and selected by the company PALL. Filters wereobtained after filtration of the CSF from a few cases of MS, and theproteins adsorbed to the filters were redissolved using a solutioncontaining 1% of SDS. After the samples were brought to physiologicalconditions, it could be shown that a strong gliotoxic activity,evaluated by the present biological test on an astrocyte line, isassociated with the proteins previously retained on these filters.

An example of the use of the biological test for detecting andquantifying gliotoxic activity in biological fluids, in particular inthe CSF of patients suffering from MS, is presented in attached Table 1,where the effects of the filtration of the CSF through the filtersupplied by the company PALL are shown.

In this table, the CSF were filtered through PALL filters having thesame type of filtration medium as those used in vivo in therapeutictrials. The gliotoxic activity of each filtrate, measured according tothe biological test on astrocytes, and the result in comparison with thegliotoxic activity of the native CSF before filtration, with thestatistical significance of the difference observed on a series of 10measurements, are presented therein. In this example, the CSF werediluted to 1/10 in the culture medium and incubated for 72 hoursaccording to the protocol described above for the "L/D test". The meanvalue of the percentage of dead cells was obtained, for each sample, on5 fields chosen at random on two wells incubated in parallel with thesame sample. A statistically significant difference exists between theMS CSF and the filtrates obtained after filtration. The diagnoses of MSare, except where otherwise stated, diagnoses of definite MS accordingto the criteria of Poser (49).

This example shows that physicochemical means exist for removing atleast part of said gliotoxic factor from the biological fluids ofpatients suffering from MS, or from other diseases in which saidgliotoxic factor or alternatively said gliotoxic activity might bedetected in vivo. These results also confirm the molecular reality ofsaid gliotoxic activity, as well as the importance of the detection andassay techniques which are subjects of the present invention.

Another example of the use of the biological test developed by theinventors, for detecting and quantifying gliotoxic activity inbiological fluids, in particular in the CSF of patients suffering fromMS, is presented in attached Table 2. In this example, the CSF werediluted to 1/20 in the culture medium according to the protocoldescribed above for the calorimetric MTT test, and incubated for 96hours. Each result represents the mean of two separate experiments eachrepresenting a series of 5 independent wells, that is to say, finally, amean of 10 values per CSF tested.

The CSF samples originating from patients suffering from a remittingform of MS were taken at the time of clinical exacerbations. Thedifference in the mean cytotoxicity values in the two subpopulations ofMS, remitting versus progressive chronic, is statistically significant,indicating in principle that the quantification of the gliotoxicactivity of the CSF enables the clinical activity of the disease to becorrelated. The diagnoses of MS are, except where otherwise stated,diagnoses of definite MS according to the criteria of Poser (49).

The results presented in Tables 1 and 2, together with other similarresults, thus enabled the inventors to demonstrate the existence of verysignificant gliotoxic activity in the CSF of patients suffering from MS,and a variation of its intensity in accordance with the clinical stateand/or the mode of progression. These results confirm the importance ofthe biological test developed on an astrocyte line, in combination witha quantification technique which can be, for example, one of the threetechniques described above ("L/D test", MTT calorimetric assay and ⁵¹Cr-release assay), in studies to determine prognosis and/or in thetherapeutic monitoring of pathologies in which such a cytotoxic, and inparticular gliotoxic, activity may be detected, or even in the diagnosisof a disease such as MS.

Example 8

Cytotoxic activity observed on other cell types.

Moreover, the specificity of the observed cytotoxic activity was alsoevaluated, and quantified by the "L/D test", on cultures of variousother cell types. With samples which had previously been shown to becytotoxic with respect to glial cells, either in culture of ratembryonic brain explants or on the astrocyte line, no directcytotoxicity was detected on fibroblasts, myoblasts and mouse leg musclecells and on endothelial cells. A markedly lower cytotoxicity, ofapproximately 10% relative to the abovementioned astrocytes, wasobserved on sciatic nerve Schwann cells. It is of interest to note herethat the Schwann cells are responsible for the myelination of peripheralnerves, just as the oligodendrocytes are responsible for normalmyelination in the central nervous system. In the cultures of embryonicbrain explants, the few leptomeningeal cells present do not appear to bedirectly affected, and the same applies to the neurons, which appear tobe affected only after considerable modification of the nutritive lawnof glial cells. Blood monocytes set up in culture and which havedifferentiated into macrophages after adhering to the culture support donot appear to be affected either, since they persist in culture even inthe presence of several successive peaks of gliotoxic activity releasedinto the supernatant in the course of macrophage culture (see FIG. 5).As regards lymphocytes, contradictory results have been obtained withcultures of cells expressing the CD4 antigen, namely, with the samegliotoxic sample compared with the same nongliotoxic control sample,significantly increased cell death was observed in some cultures,whereas a cell proliferation was observed in cultures originating fromother lymphocyte-donor individuals.

These observations on lymphocytes have similarities with the effectsdescribed for molecules having superantigenic properties (26, 27).

Example 9

Cytological characterization of the cytotoxic effects.

These data obtained with lymphocytes led the inventors to clarifybeforehand the cytological effects linked to the cytotoxicity, on theastrocytes used in the biological tests for gliotoxicity. A study of theintermediate filaments of the abovementioned astrocytes revealed aconsiderable effect of gliotoxic fluids on the organization of theastrocyte cytoskeleton, after incubation of the cells according to theprotocol described above for the gliotoxicity tests. In effect, whereasno modification is observed with the nongliotoxic control fluids testedin parallel, a drastic disorganization of the vimentin and GFAPfilaments is observed in the cultures of astrocytes brought into contactwith significant gliotoxic activity originating from biological fluidsfrom patients suffering from MS (monocyte/macrophage culture or CSF).This phenomenon had also been observed on the primary cultures ofembryonic nervous system explants, in which the astrocytes werespecifically labeled with an anti-GFAP antibody.

An example of this disorganization of the vimentin and GFAP intermediatefilaments on astrocytes in cultures brought into contact with gliotoxicfluids originating from patients suffering from MS is presented in FIG.7. In this example, the detection of GFAP was performed on a primaryculture of cells cultured on Labtek® type slides. After incubation forone hour and 24 hours with the culture medium containing a 1/20 dilutionof a gliotoxic MS monocyte/macrophage culture supernatant, the adherentcells on the slide are rinsed twice in PBS and then fixed in 4%paraformaldehyde for 20 min at 37° C. After 2 rinses in PBS, they areincubated for twice 5 minutes in a PBS/1% milk blocking solution. Thefirst antibody is polyclonal, raised in rabbits. The antibody is dilutedto 1/50 in the blocking solution. Incubation lasts 1 h at 37° C. Afterthe first incubation, a series of 5-minute rinses in PBS and then in theblocking solution is performed in order to remove unbound antibodies.The visualization system employs fluorescein coupled to an anti-rabbitimmunoglobulin diluted to 1/100 in the blocking solution. Incubationlasts 1 h at 37° C. Mounting of the preparations is carried out withmoviol in order to avoid a decrease in fluorescence at the time ofobservation. The slides are stored in darkness at 4° C. Observations aremade with a fluorescence microscope. The detection of vimentin wasperformed on the astrocyte line cultured on Labtek® type slides, afterincubation for 24 hours with either a 1/50 dilution of a gliotoxic MSmonocyte/macrophage culture supernatant, or a normal culture medium.After 24 hours of incubation, the cells (1×10³ cells/cm²) are rinsedtwice in PBS and then fixed for 10 min at -20° C. with acetone. After 3rinses in PBS, they are incubated for three times 5 minutes in a PBS/5%FCS blocking solution at room temperature. The first antibody ispolyclonal, raised in goats. The antibody is diluted to 1/50 in theblocking solution. Incubation is for 2 h at 37° C. After 3 5-minuterinses in PBS and then in the blocking solution, the cells are incubatedfor 1 h at 37° C. with a second antibody directed against goat IgGs,coupled to fluorescein and diluted to 1/200 in the blocking solution.Photograph sic! A and B show a labeling of the GFAP after 1 and 24 hoursincubation, respectively, in the presence of the diluted gliotoxicsample. Photographs C and D show a labeling of the vimentin of theastrocytes in the line, incubated for 24 hours with a normal medium anda medium containing the diluted gliotoxic sample, respectively.

Furthermore, the inventors studied the appearance of the cellular DNAextracted from cultures of astrocytes cultured in normal medium andcultured for increasing times in the presence of gliotoxic samplesoriginating from MS. It was then found that the DNA originating fromcultures subjected to gliotoxic activity displayed a fragmentation ofthe cellular DNA, the intensity of which increased in accordance withthe incubation time of the cells, whereas the DNA of cells incubated inmedium without gliotoxic activity remained homogeneous at a very highmolecular weight.

These observations are compatible with an apoptosis process such as maybe induced by superantigens, on lymphoid cells in particular.

Example 10

Dose-response effect

Subsequently, in order to evaluate the possibility that this gliotoxicactivity detected is borne by a molecule, or by a factor whose molecularrepresentation might prove more complex, present in the fluids tested,the inventors first evaluated, using the biological tests developed anddescribed above, the reality of a dose-response effect on thecytotoxicity induced by reference samples. A dose-response effectcompatible with a direct proportionality of cell death with respect tothe dilution, and with an effect of saturation of the astrocytedetection system at high concentrations, was observed aftervisualization of the effect by the "L/D test".

An example of this dose-response effect observed with a gliotoxicbiological fluid is presented in FIG. 8. In this example, an MSmonocyte/macrophage culture supernatant was mixed, in successivedilutions, with the culture medium of the astrocytes according to theprotocol described above for the "L/D test".

In this way, the inventors characterized a cytotoxic, and moreespecially a gliotoxic, activity in the cultures of bloodmonocytes/macrophages, CSF and serum of patients suffering from MS, inparallel with the development of a method for detecting said cytotoxicactivity on primary cultures of brain and spinal cord explants of ratembryos, and said gliotoxic activity on astrocyte lines in culture, aswell as also the development of a method for quantifying said gliotoxicactivity, coupled to the detection method on astrocytes in culture.

Their first objective having been the demonstration of this gliotoxicactivity in vivo in patients suffering from MS, and the means for itssystematic and standardized assay, the inventors then directed theirattention to endeavoring to link this activity with a particularmolecule, or a factor represented by a set of defined molecules, in thefluids tested.

Example 11

Characterization of the cytotoxic/gliotoxic factor

The inventors first observed that the biological activity defined asgliotoxic activity by the methods described above persisted in gliotoxicsamples placed in a water bath at +56° C. for half an hour but wasabolished when the temperature was +100° C. for 15 minutes, andpersisted after freezing the sample to -80° C. followed by thawing to+37° C. After centrifugation at 100,000 g for 2 hours and removal of thepellet of sedimented material, the activity of samples previouslyrecognized as gliotoxic according to the methods described in thepresent invention was still present in the supernatant, and henceresembles a nonparticulate soluble factor. Similarly, the incubation ofgliotoxic samples in which the total amount of protein has previouslybeen determined by the technique of Bradford (53) in the presence oftrypsin, pronase, proteinase K or a mixture of N-glycosidase F andneuraminidase, under sufficient conditions for complete enzymatichydrolysis of the peptide bonds or of the N-glycosylations present inthe sample, does not abolish their gliotoxic activity as is describedabove.

Subsequently, the inventors carried out the separation and fractionationof the different compounds from samples which had previously provedgliotoxic by the methods described in the present invention. For all ofthese investigations, the samples are previously heated for 30 min at56° C. and centrifuged for 10 min at 1500 rpm, and the supernatant isthen recovered and, where appropriate, dialyzed at 4° C. in twice 20volumes of D-PBS buffer, the first time for 2 h and a second timeovernight. The supernatant thus collected constitutes the sample onwhich the different operations are performed. Furthermore, the fractionsoriginating from samples whose molecular components have been separatedby different methods are treated so as to remove possible toxicmolecules introduced into the media collected by the methods used, andso as to redissolve the organic molecules under physiological conditionscompatible with the methods for detecting and quantifying the gliotoxicbiological activity as are described in the present invention. To thisend, the samples are lyophilized, resuspended in 2.5 ml of steriledistilled water, applied to an NAP-25 type chromatography column(Pharmacia) previously washed in 10 volumes of D-PBS buffer, then elutedwith 3.5 ml of D-PBS buffer and used as they are for the gliotoxicactivity test.

Thus, to study the ionic charge of the gliotoxic factor as definedabove, samples which were gliotoxic according to the criteria definedabove and originating from MS monocyte/macrophage culture supernatantsand CSF were passed at a flowrate of 60 ml/hour through an FPLCchromatography column of the DEAE-Sepharose CL-6B (Pharmacia) typeequilibrated in a buffer A (50 nM Tris-HCl, pH 8.8). Under theseconditions, the fraction bearing gliotoxic activity present in theoriginal sample is eluted with an ionic strength of between 0.12M and0.2M NaCl of buffer A.

To study a possible physicochemical analogy with certain serineproteases which have a strong affinity for liquid chromatographysupports of the Blue-Sepharose (Pharmacia) type, samples which weregliotoxic according to the criteria defined above, originating from MSmonocyte/macrophage culture supernatants and CSF and previously dialyzedat 4° C. in twice 20 volumes of buffer B (50 mm Tris, pH 7.2) andcontaining 5 mg of proteins per ml, were applied to an FPLC column ofthe Blue-Sepharose CL-6B (Pharmacia) type. After elution with 0.1M KClin buffer B, no gliotoxic activity was to be found in the eluate,whereas the fraction bearing gliotoxic activity was recovered with anactivity yield close to 70% by elution with 1.5M KCl in buffer B.Analysis also revealed that serum albumin was also eluted in this samefraction. However, the protease activity of the eluate was tested byincubating the latter with "azocasein" (Sigma) or "azocoll" (Sigma) inD-PBS buffer, pH 7.5 at 37° C. for 2 hours, without it being possible todemonstrate any proteolytic activity of the sample thus prepared andeluted.

To study the possible association of such a gliotoxic factor with IgGtype immunoglobulins, samples which were gliotoxic according to thecriteria defined above and originating from MS monocyte/macrophageculture supernatants and CSF were applied to an FPLC column of theprotein A-Sepharose CL-4B (Pharmacia) type previously washed with 5volumes of D-PBS. The protein A content of the swollen gel was equal to2 mg/ml, and the capacity for binding human IgG was of the order of 20mg of IgG/ml of gel (gel volume 1.5 ml). The fraction lacking IgG wasrecovered by elution with D-PBS and contained the gliotoxic activity,whereas the fraction enriched in IgG, eluted with 50 mm sic! glycine-HClbuffer, pH 3.0, did not contain gliotoxic activity as defined above whenthe samples originated from CSF or from supernatants ofmonocyte/macrophage cultures performed under the conditions describedabove. In the case of samples originating from human sera, a lowgliotoxic activity was to be found in the fraction enriched in IgG, thisbeing the case both for sera from MS in exacerbation drawn at the sametime as a CSF which proved gliotoxic, and for sera from controlssuffering, for example, from normal pressure hydrocephalus (NPH), whoseCSF drawn at the same time did not display any gliotoxic activity.However, the study of NPH or of healthy control sera did not reveal anygliotoxic activity in the fraction lacking IgG eluted with D-PBS,whereas almost the whole of the considerable gliotoxic activity of thesera from patient sic! suffering from MS in exacerbation was to be foundin this fraction lacking IgG eluted with D-PBS. This suggests that thereis/are one or more gliotoxic component(s) in the serum of healthyindividuals which are different from the gliotoxic factor which has beendemonstrated by the inventors of the present invention, of markedlylower activity and which is to be found eluted in the fraction enrichedin IgG after passage through a protein A-Sepharose column under theconditions described above. This probably resembles a nonspecificgliotoxicity linked to active proteins of serum, as has already beenreported (54). However, the small amount of human serum originating fromhealthy donors having an AB positive blood group present in themonocyte/macrophage cultures does not appear to produce, under theseanalytical conditions, this additive gliotoxicity collected in this waywith the elution of IgG. This may be due to the dilution effect which,as a result of the low activity of this component, renders itundetectable by our methods, or alternatively to an effect ofinactivation of this component during the culturing ofmonocytes/macrophages under the conditions described above. If there isadded to this fact the absence of detectable gliotoxicity under theseconditions after one and the same analysis of nongliotoxic samplesoriginating from CSF of controls suffering from NPH or originating fromculture supernatants of monocytes/macrophages from healthy individuals,these results confirm the novel nature of the gliotoxic activity, andthereby of the associated gliotoxic factor, constituting subjects of thepresent invention, relative to the gliotoxic activities capable ofoccurring in the absence of a pathological process in the biologicalfluids of apparently healthy individuals.

To study the molecular weight of said gliotoxic factor, the inventorsconcomitantly and successively analyzed samples which were gliotoxicaccording to the criteria defined above, and originating frommonocyte/macrophage culture supernatants, from PALL filters which wereused for filtering in vivo the CSF of MS patients and from MS CSF, on anFPLC column of the Superose 12 (Pharmacia) type and on polyacrylamidegel electrophoresis in the presence of SDS (SDS-PAGE). After analysis ona Superose 12 column, the fractions containing gliotoxic activity wereeluted after an elution volume corresponding to a molecular weight ofapproximately 17 KD by matching against the reference curve of theelution volumes of standard globular proteins. More precisely, aftercollection of fractions which were closer together in this elution zone,two separate peaks of gliotoxicity could be observed, at elution volumescorresponding to a molecular weight of approximately 21 KD and ofapproximately 16.8 KD. A similar elution of the same sample in a bufferto which 8M urea has been added gives identical results, indicating thatthere are no multimeric associations of the components eluted underphysiological buffer conditions.

After analysis by one-dimensional SDS-PAGE, the protein bands containinggliotoxic activity, after elution from the gel and a return tophysiological conditions, were to be found in a portion of gelcorresponding to a molecular weight of approximately 17 KD and, undercertain conditions of analysis and of content of biological materialanalyzed, in an additional portion of approximately 21 KD. In parallel,the other protein bands as well as different regions of the gel withoutproteins were tested, without it being possible to detect anysignificant gliotoxic activity therein. Two-dimensional analysis of thegliotoxic protein band extracted from the gel at around 17 KD showed theexistence of two major spots at 17 KD displaying, between approximatelypH 6 and pH 7, a different isoelectric point, and a minor spot at 18 KDhaving a slightly more basic isoelectric point, above a pH ofapproximately 7.

An example of analysis by one- and two-dimensional gel electrophoresisof the protein components of a gliotoxic fraction obtained after passagethrough a DEAE column is presented in FIGS. 9A and 9B. In FIG. 9A, thephotograph may be seen of a Coomassie blue-stained one-dimensionalacrylamide gel originating from a mixture of gliotoxic samples elutedfrom some ten PALL filters which were used for the in vivo filtration ofthe CSF of a series of patients suffering from MS. These filters areopened mechanically and the filters washed in the presence of 1% SDS,and the eluates are then returned to physiological buffer by means, inparticular, of a passage through an NAP-25 column as described above.The filtrates thus treated are tested for their gliotoxic activity on analiquot, and passed as a mixture through a DEAE-Sepharose column. Thefraction containing gliotoxic activity is desalted on an NAP-25 columnand concentrated by evaporation before being applied in two parts, oneto a one-dimensional SDS-PAGE gel (FIG. 9A) and the other to atwo-dimensional SDS-PAGE gel. A photograph of such a two-dimensional gelis presented in attached FIG. 9B. The band visualized with an apparentmolecular weight of 17 KD in one dimension proves to be the only proteinband visualized bearing gliotoxic activity on the gel shown in 9A, andit may be seen on the two-dimensional gel (9B) that this band ofapproximately 17 KD separates into three spots of different isoelectricpoints, the third spot on the right having an apparent molecular weightslightly higher than the two spots at the left.

An example of analysis with a Superose 12 FPLC column is presented inFIGS. 10A and 10B. In this example, culture supernatants ofmonocytes/macrophages from a patient suffering from MS, sampled betweenthe 6th and the 16th day of culture, representing a volume of 20 ml and140 mg of proteins, were first passed at a flow rate of 60 ml/hourthrough an FPLC chromatography column of the DEAE-Sepharose CL-6B(Pharmacia) type equilibrated in a buffer A (50 mM Tris-HCl, pH 8.8).Concomitantly, a Superose 12 column was equilibrated with a buffer C, 50mM Tris-HCl, pH 6.8, and calibrated by eluting a mixture of globularproteins of known molecular weight in this same buffer C. The fractiontheoretically bearing gliotoxic activity present in the original samplewas eluted from the DEAE-Sepharose column with an ionic strength between0.12M and 0.2M NaCl of buffer A, in a volume of 15 ml containing 39 mgof proteins. One third of this fraction, containing 13 mg of proteins,was then applied to the Superose 12 column in a buffer without urea(FIG. 10A), and another third to an identical column in 8M urea buffer(FIG. 10B). During elution in buffer C, 40 fractions were collected, anda continuous measurement of the absorption at 280 nm to assay theproteins in the eluate was recorded with a high sensitivity of detection(R=1.2). The gliotoxic activity of the different fractions collected wastested according to our biological test on an astrocyte line andquantified, after 72 h of incubation, by the "L/D test" technique. Tothis end, the fractions were lyophilized, resuspended in 2.5 ml ofsterile distilled water, applied to an NAP-25 (Pharmacia) typechromatography column previously washed in 10 volumes of D-PBS buffer,then eluted with 3.5 ml of D-PBS buffer and used as they were for thegliotoxic activity test. In FIGS. 10A and 10B, the curve in a continuousheavy line represents the optical density at 280 nm, and the curve in abroken line represents the gliotoxic activity. The correspondencesbetween the elution volume (Ve) and the apparent molecular weight werecalculated in accordance with the calibration carried out previouslywith reference proteins, and is noted at the top of the peaks ofgliotoxic activity. It should be noted that a slight gliotoxic activityis to be found in a fraction corresponding to a molecular weight ofapproximately 110 KD. However, the similarity of the elution profiles inthe presence and absence of 8M urea supports the view of a monomericcomposition of the factor eluted at these different molecular weights.Accordingly, in this example, the gliotoxic activity specific to thefactors of apparent molecular weights approximately 21 KD andapproximately 17 KD can be demonstrated and matched to the individualfactors.

The results obtained by FPLC chromatography on a Superose 12 column andby electrophoretic analysis show that the gliotoxic factor consists atleast of a protein or associated molecule of molecular weight 17 KD anda protein or associated molecule of molecular weight 21 KD. The factthat the apparent molecular weights at which the gliotoxic activity isto be found are identical in both types of technique (FPLC andelectrophoresis) suggests that the factors in question are globularproteins. Furthermore, since a comparative study of SDS-PAGE gels in thepresence or absence of β-mercaptoethanol gave, moreover, identicalmigration profiles, these proteins are probably homomers. The fact thatthe gliotoxic activity is to be found, among all the chromatographyfractions and all the portions of gel analyzed, at two differentmolecular weights of approximately 17 KD and approximately 21 KD may beexplained by two molecules without significant homology, or by theexistence of glycosylations or of any different post-translationalmodifications on the same protein substrate, or alternatively by theexistence of a propeptide or of a peptide portion of some kind which,after cleavage of a protein of approximately 21 KD, generates a proteinof approximately 17 KD.

Thus, to study the possible glycosylation of said gliotoxic factor,samples which were gliotoxic according to the criteria defined above andoriginating from MS monocyte/macrophage culture supernatants, sera andCSF and from filters which were used for filtering in vivo the CSF of MSpatients were analyzed on an FPLC column of the concanavalin A-Sepharose(Con A-Sepharose, Pharmacia) type. For this study, the gliotoxicbiological examples were previously passed through an FPLC column of theDEAE-Sepharose type or of the protein A-Sepharose type. The fractionscontaining gliotoxic activity, that is to say a fraction eluted at about0.2M NaCl and a fraction eluted with D-PBS, respectively, are then usedfor FPLC chromatography on a Con A-Sepharose type column. Under theseconditions, the fractions eluted from the Con A-Sepharose column, eitherwith D-PBS containing up to 500 mM NaCl or thereafter with 200 mMD-glucopyrano-side which displays a competitive affinity for Con A, didnot display any gliotoxic activity. The gliotoxic activity present inthe original sample was to be found concentrated in the fraction eluted,after the two elutions mentioned above, with 50 mM glycine-HCl buffer,pH 3.0 with 1 mM Ca⁺⁺ and 1 mM Mn⁺⁺. This fraction corresponds tomolecules having a high affinity for Con A, which corresponds in generalto strongly glycosylated molecules. Analysis of this fraction inone-dimensional SDS-PAGE reveals that the gliotoxic activity is to befound associated with two protein bands of 17 KD and 21 KD,respectively.

An example of analysis on one-dimensional SDS-PAGE gel of a purificationusing an FPLC column of the DEAE-Sepharose Con A-Sepharose type ispresented in FIG. 11. In this example, 27 ml of supernatant containing100 mg of proteins, from a culture of monocyte/macrophage from a patientsuffering from MS, were passed through a DEAE-Sepharose column. sic! andthe eluate obtained with an ionic strength of between 0.12 and 0.20MNaCl was collected in a volume of 10 ml containing 32 mg of proteins.This fraction was then passed through a Con A-Sepharose type column. Afirst elution was carried out with 500 mM D-PBS, a second elution wascarried out with D-PBS buffer containing 200 mM D-glucopyranoside, and athird elution was carried out with 50 mM glycine-HCl buffer, pH 3.0 with1 mM Ca⁺⁺ and 1 mM Mn⁺⁺, and an eluate of 6.4 ml containing 0.17 mg ofproteins was collected.

After this series of purifications in which the gliotoxicity of all theintermediate samples was assayed, 79% of the gliotoxicity of theoriginal supernatant was to be found in the fraction eluted in glycinebuffer, pH 3 from the Con A-Sepharose column, with a proteinpurification yield of 465 times.

On an SDS gel containing 10% of acrylamide, samples from each step wereapplied successively to parallel wells, corresponding to an amount ofprotein of 2.2 mg for the third fraction eluted on Con A-Sepharose. Itshould be noted that still better yields can be obtained for the samesteps with CSF from patients suffering from MS.

Furthermore, starting from the third fraction eluted on Con A-Sepharose,a preparative SDS-PAGE electrophoresis was performed in parallel, anddifferent bands were cut out from the unstained gel at the distancecorresponding to all the protein bands stained with Coomassie blue in areference well, as well as from a few regions without protein. Toanalyze the gliotoxicity of each band thus cut out, the pieces of gelwere crushed and homogenized in D-PBS containing 0.2% of SDS, incubatedat 37° C. for 30 minutes and then centrifuged at 100,000 g for 6minutes. The operation is repeated twice, and the supernatants of thetwo centrifugations are mixed and passed through a DEAE-Sepharosecolumn. The eluate obtained by elution with D-PBS 200 mM NaCl buffer iscollected and used for the gliotoxicity tests. Under these conditions,the SDS is retained on the column and the eluate is physiologicallycompatible with the cell cultures. In these electrophoresis gelextracts, significant gliotoxic activity was to be found only in thebands cut out in the molecular weight regions of approximately 17 KD and21 KD. In order to verify the protein profile of these two extracts,they were applied to the analytical gel in parallel with the abovechromatography fractions. In FIG. 11 presenting the results of thevisualization of the proteins which migrated after electrophoresis,starting from the left, the first column shows a series of standardproteins of known molecular weights which are indicated on the left, thesecond column under "S" shows the starting supernatant, the third columnunder "I" shows the fraction eluted at between 0.12 and 0.2M NaCl onDEAE-Sepharose, and the fourth, fifth and sixth columns under "II" showthe three successive elutions performed on a Con A-Sepharose column andare headed by the serial numbers 1, 2 and 3, respectively, in the orderof the above description. The last two columns correspond to the twogliotoxic bands separated and extracted from a preparative SDS-PAGE gel.

From these results it emerges that, as in the analysis performed on MSbiological fluids on a superose 12 column and in one-dimensionalSDS-PAGE starting from eluates of filters used for the in vivofiltration of MS CSF, the gliotoxic activity is to be found associatedwith proteins of apparent molecular weight of approximately 17 KD and 21KD, with a markedly lower concentration for the 21 KD molecule. However,and contrary to the observations made previously, nongliotoxic controlsamples passed through a Con A-Sepharose column under the sameconditions displayed these bands at 17 KD and 21 KD on SDS-PAGE gel inthe fraction eluted in glycine-HCl buffer. Evaluation of the gliotoxicactivity of these control samples under the conditions described abovedoes not, however, reveal any significant cytotoxic effect at any stepof purification, nor does it do so in the protein bands at approximately17 KD and approximately 21 KD. Furthermore, "blank" elution of a ConA-Sepharose column with the abovementioned pH 3 glycine-HCl bufferenables the same nongliotoxic bands to be visualized. The possibility ofa detachment of protein subunits or fragments of Con A from thechromatography support by the pH 3 glycine buffer was verified. However,in order to verify the reality of a comigration of a novel moleculebearing gliotoxic activity occurring in the bands of approximately 17and 21 KD originating from MS samples eluted on Con A-Sepharose underthe abovementioned conditions, the inventors carried out a digestion,under appropriate conditions, with proteinase K of the fractionoriginating from a gliotoxic sample and of a nongliotoxic control sampleeluted under the same conditions on Con A-Sepharose with the glycine-HClbuffer. The two digestion products were then eluted in parallel on aSuperose 12 FPLC column under the conditions mentioned above. Theseanalyses enabled it to be demonstrated that, in the eluate originatingfrom the "MS" sample, an undigested protein peak associated withgliotoxic activity and free from protease activity was always present,whereas all the protein material was degraded in the fractionsoriginating from nongliotoxic control samples previously containing 17-and 21-KD proteins.

An example of this analysis is presented in FIGS. 12 and 13. In thisexample, a mixture of MS monocyte/macrophage supernatants displayingsignificant gliotoxic activity and containing 3 g of proteins and anequivalent sample originating from a control culture without significantgliotoxic activity were passed in parallel through a DEAE-Sepharose FPLCcolumn, the fractions eluted at between 0.12 and 0.2M NaCl recovered andthe equivalent of 2 mg of proteins for each sample passed through a ConA-Sepharose FPLC column. The fractions eluted in 50 mM glycine-HClbuffer, pH 3 with 1 mM Ca⁺⁺ and 1 mM Mn⁺⁺ were first redissolved in anappropriate buffer. To this end, the samples were lyophilized,resuspended in 2.5 ml of sterile distilled water, applied to an NAP-25(Pharmacia) type chromatography column previously washed in 10 volumesof 20 mM Tris-HCl buffer, pH 8.0, 1 mM Ca⁺⁺, 0.1% SDS, then eluted withthe same buffer and used as they were for incubation with proteinase K.The enzyme used is immobilized (Proteinase K-Acrylic beads, Sigma ref.P0803) and used in a ratio of 20 mU/50-100 μg of protein in 20 mMTris-HCl buffer, pH 8.0 with 1 mM Ca⁺⁺ and 0.1% SDS. The samples to bedigested were incubated for at least 16 hours at 37° C. The supernatantsrecovered after centrifugation and sedimentation of the beads coupled toproteinase K were then passed through a Superose 12 FPLC column underthe conditions described above, and eluted with 50 mM Tris-HCl buffer,pH 6.8 containing 8M urea in order, in particular, to disassociate anymultimeric protein. FIG. 12 shows the elution on Superose 12 of thegliotoxic sample originating from MS after treatment with proteinase K.FIG. 13 shows the elution on Superose 12 of the nongliotoxic sampleoriginating from controls not suffering from MS after treatment withproteinase K. The curves in a continuous heavy line show the absorptionof the eluate at 280 nm, with an optical density measurement ofsensitivity R=0.02, indicating the relative protein concentration. Thepoint of the curve corresponding to the elution of globular proteins ofapproximately 17 KD is indicated by an arrow. A protein peak ofapproximately 17 KD is visible only in FIG. 12, and is associated with agliotoxic activity evaluated at 75% cytotoxicity on a dilution of thesample to 1/10, according to the biological test according to theinvention, after 72 h of incubation and quantification of relative celldeath by the L/D test. Moreover, in order to detect any trace ofcontaminant proteinase K, a test of protease activity on azocoll (Sigmaref. A9409) was performed, and did not enable any contaminant protein tobe detected at this level. In contrast, a similar absorption peak at 280nm is detected in both samples below 5 KD and corresponds to theproducts of degradation of digestible proteins by proteinase K.

The results presented in the example illustrated by FIGS. 12 and 13 showthat there is indeed a specific gliotoxic factor present in a novelmanner in the biological fluids originating from patients suffering fromMS, the factor being associated with one or two polypeptide molecule(s),at least one (or more) 17 KD region(s) of which cannot be digested byproteinase K. However, the mode of purification using Con A-Sepharosecolumns, while it has enabled a very strong affinity of the gliotoxicfactor for concanavalin A to be demonstrated, does not enableappropriate samples to be obtained for a subsequent peptide analysis, asa result of a contamination by components of the concanavalin Aoriginating from the columns under the elution conditions required foreluting said gliotoxic factor. Under these conditions, it seems that apurification protocol successively combining a DEAE-Sepharose column anda Superose 12 column would be best suited to a strategy of purificationof molecules bearing the specific gliotoxic activity characterized inthe present invention.

An example of analysis of the purification yield with different FPLCcolumns is presented in Table 3.

In this example, the cytotoxicity is expressed as the amount of protein(mg) needed to have 50% cell death, on the basis of a quantification bya methyltetrazolium colorimetric assay after incubation at 37° C. for 72h of the test sample diluted to 1/10 in the culture medium. Thefractions tested are, for each column type, those which are eluted underthe conditions previously described for containing the gliotoxicactivity. The yield is calculated according to the following formula:##EQU1##

bp: before purification; ap: after purification.

In this example, it is apparent that protein A-Sepaharose sic! columnscould also be used advisedly, prior to another method of separationexcluding Con A-Sepharose columns, in a strategy of purification of thegliotoxic factor.

Lastly, after studying different techniques of purification andpreparation of molecular fractions associated with the gliotoxicactivity demonstrated in the biological fluids originating from patientssuffering from MS, the inventors verified in the study governing thepresent invention that the purification, even partial, of moleculesconstituting the molecular basis of the gliotoxic activity affords adose-response effect which is even more clearly definable than on thecrude fluids previously tested, this being done, in particular, in orderto verify the biopharmacological reality of the purification of saidgliotoxic factor.

The two examples which follow illustrate the reality of a purificationof a biopharmacological activity in parallel with the molecularpurification performed.

In the example illustrated by attached FIG. 14, for which the curveshave been plotted from the data presented in the attached Table No. 4, aseries of dilutions was made in PBS buffer with a culture supernatant ofmonocyte/macrophage from a patient suffering from MS (MS 1), and saiddilutions were incubated for 72 hours with a final dilution ranging from1/20 to 1/5000 in the culture medium of wells comprising a monolayer ofastrocytic cells originating from the astrocyte line described above. Afraction of the same MS monocyte culture supernatant, obtained afterpurification of the gliotoxic fraction by passage through a ConA-Sepharose column, was diluted and incubated in parallel according tothe same protocol. The astrocytic cells were incubated, immediatelybefore the introduction of the gliotoxic dilutions, with chromium-51,and washed in order to remove radioactive isotopes not incorporated intothe living cells. After 72 hours of incubation with the two series ofdiluted samples, the radioactivity released into the supernatant ismeasured with a gamma counter and compared with that measured in thecells remaining in culture at the bottom of the wells. The percentagecytotoxicity, as explained above, represents the proportion ofradioactivity released into the supernatant by the dead cells. It maythus be seen in FIG. 14 that there is a dose-response effect marked by agradual decrease in the cytotoxicity measured as a function of theincreasing dilutions of the sample. It should, however, be noted thatthe slope of the curve is markedly increased, over the same dilutionintervals, with the purified factor. This confirms the reality of thepurification and of the molecular concentration of the factor associatedwith the biological activity measured by our test of gliotoxicity.

In the example illustrated by attached FIG. 15, for which the curveswere plotted from the data presented in attached Table 5, a series ofdilutions was made in PBS buffer with a culture supernatant ofmonocyte/macrophage from a patient suffering from MS (MS 1), and saiddilutions were incubated for 72 hours with a final dilution ranging from1/20 to 1/5000 in the culture medium of wells comprising a monolayer ofastrocytic cells originating from the astrocyte line described above. Afraction of the same MS monocyte culture supernatant, obtained afterpurification of the gliotoxic fraction by passage through a ConA-Sepharose column, was diluted and incubated in parallel according tothe same protocol. After 72 hours of incubation with the two series ofdiluted samples, cells remaining viable in the culture wells aredetected by the methyltetrazolium (MTT) test described above, and thecolored product generated by the functional mitochondrial enzymes ofthese cells is assayed by measuring the optical density of thesupernatant at between 570 and 630 nm. The optical density, as explainedabove, represents the amount of living cells remaining in each wellpreviously inoculated with the same number of cells maintained in aconfluent monolayer at the bottom of the culture well in a survivalmedium. It may thus be seen in FIG. 15 that there is indeed adose-response effect marked by a gradual increase in cell survivalmeasured as a function of the increasing dilutions of the sample. Itshould, however, be noted that the slope of the curve is markedlyincreased, over the same dilution intervals, with the purified factor.This further confirms, by this technique of quantification of livingcells, the reality of the purification and of the molecularconcentration of the factor associated with the biological activitymeasured by our test of gliotoxicity.

In the last two examples illustrated by FIGS. 14 and 15, two assaytechniques measuring opposite parameters, such as cell survival andrelative cell mortality, achieve entirely concordant results and, whileenabling the reality of a purification of the gliotoxic activitydetected at the same time as gliotoxic factor to be demonstrated, showclearly the discriminatory power and the reliability of the biologicaltest forming, together with the gliotoxic factor characterized by theauthor's investigations, the subject of the present invention.

Thus, on the basis of the discovery of a cytotoxic activity capable ofbeing demonstrated in vivo in individuals suffering from MS andpreferentially targeting the glial cells, a method for detecting andquantifying the cytotoxic, and more especially gliotoxic, activityassociated with this factor has been invented, developed and validatedunder different conditions of use. Furthermore, said method has made itpossible to study and characterize the molecular basis of this gliotoxicactivity, in the form of protein fractions having two apparent molecularweights of approximately 17 KD and approximately 21 KD, in biologicalfluids originating, in particular, from patients suffering from MS. Thefact that there is a portion which cannot be digested by proteinase Kunder non-denaturing conditions which is to be found associated with thegliotoxic activity at 17 KD by elution on a Superose 12 FPLC columnpossibly suggests that an additional peptide which can be digested(propeptide for example) differentiates the 21-KD form from that of 17KD. These two factors, apparently of the protein type, of approximately17 and approximately 21 KD are probably globular, without disulfidebridges connecting independent peptide chains, somewhat hydrophilic,negatively charged at neutral pH and apparently glycosylated despite thefact that incubation in the presence of N-glycosidase F andneuraminidase does not abolish their gliotoxic activity. Furthermore,they display a very high affinity for at least one lectin, concanavalinA or Con A. These protein factors have a biological activity whichwithstands incubation at 56° C. for half an hour and which disappearsafter heating at 100° C. for 15 minutes. This biological activity, likethe 17-KD form, withstands the action of proteases such as pronase,trypsin and proteinase K.

The detailed description set forth above has finally enabled a gliotoxicfactor possessing the following features, taken independently, to bedemonstrated:

it possesses cytotoxic activity with respect to glial cells,

this cytotoxic activity with respect to glial cells is associated withat least one globular glycoprotein,

its activity is linked to at least two protein fractions, associated orotherwise, having an apparent molecular weight of 17 KD and of 21 KD,respectively, each of these fractions possessing gliotoxic activity, the17-KD fraction being incapable of digestion by pronase or trypsin orproteinase K, and each of these two fractions displaying a strongaffinity for lectins such as concanavalin A,

the gliotoxic activity of the factor present in a sample persists afterheat treatment of the sample at +56° C. for 30 minutes, or afterfreezing the sample at -80° C. followed by thawing to +37° C.,

the factor is water-soluble and nonparticulate,

the gliotoxic activity of the factor present in a sample persists aftertreatment of the sample with trypsin or pronase or proteinase K, or amixture of N-glycosidase F and neuraminidase, under nondenaturingconditions,

the factor characterized by one-dimensional SDS-polyacrylamide gelelectrophoresis can display two bands, of 17 KD and 21 KD,

the gliotoxic factor is retained with a strong affinity on lectinsupports,

the factor is eluted at pH 7.5 in buffered 100 mM NaCl on DEAE resin,

it is not retained by protein A coupled to a support, and can hence bedifferentiated from IgG,

it is to be found in MS monocyte/macrophage culture supernatants and MSCSF and sera,

it causes a cytotoxic effect which can be quantified on astrocytic cellsin culture and can be characterized by an early effect ofdisorganization of the network of intermediate filaments, usuallyfollowed by cell death.

                  TABLE 1    ______________________________________    EXAMPLE OF APPLICATION OF THE BIOLOGICAL TEST    FOR GLIOTOXICITY    Filtration of cerebrospinal fluids from patients    suffering from MS with PALL filters                Percentage of dead cells                (L/D test)      Signifi-    Patient           Before fil-                                 After fil-                                          cance    No.   clinical stage                      tration    tration  (t-test)    ______________________________________    1     Exacerbation                      14.4 ± 1.7                                 1.4 ± 1.7                                          2.49.sup.-10    2     Exacerbation                      35.6 ± 4.55                                 9.7 ± 3.6                                          1.38.sup.-11    3     Exacerbation                      20.3 ± 3.59                                 3.2 ± 2.2                                          1.93.sup.-6    4     Exacerbation                      29.9 ± 3.28                                 9.7 ± 2.0                                          4.13.sup.-9    5     Exacerbation                      45.0 ± 3.46                                 4.2 ± 2.7                                          4.16.sup.-9    6     Chronic     20.8 ± 4.13                                 19.6 ± 2.41                                          5.15.sup.-1 NS    7     Chronic     15.5 ± 2.55                                  0.1 ± 0.32                                          9.62.sup.-9    8     Probable Clin.                      24.1 ± 3.67                                  0.6 ± 0.84                                          5.55.sup.-9    9     Exacerbation                      30.4 ± 4.97                                  4.1 ± 2.38                                          1.46.sup.-8    10    Chronic     24.3 ± 2.31                                 0.6 ± 0.7                                          3.87.sup.-9    11    Chronic     16.0 ± 2.49                                  3.3 ± 2.91                                          9.00.sup.-9    12    Exacerbation                      34.9 ± 3.75                                  0.3 ± 0.68                                          4.20.sup.-9    13    Exacerbation                      10.5 ± 2.55                                 0        3.86.sup.-7    14    Exacerbation                       0.6 ± 0.84                                 0        5.10.sup.-2 NS    15    Chronic     19.7 ± 2.21                                  0.4 ± 0.97                                          3.40.sup.-8    16    Chronic stable                      34.5 ± 1.72                                 4.8 ± 2.1                                          3.72.sup.-9    17    Chronic     23.7 ± 2.44                                 0        4.66.sup.-5    18    Exacerbation                      37.9 ± 1.88                                 2.77 ± 1.55                                          3.98.sup.-8    19    Exacerbation                      33.7 ± 2.76                                 1.34 ± 1.98                                          8.76.sup.-6    20    Exacerbation                      29.5 ± 2.98                                 2.76 ± 2.21                                          7.45.sup.-5    21    Chronic     24.8 ± 3.76                                 3.21 ± 1.55                                          2.66.sup.-7    22    Chronic     24.9 ± 2.88                                 2.54 ± 2.12                                          3.11.sup.-8    ______________________________________     * Mean of the cells in 5 microscopic examination fields chosen at random     in 2 duplicate wells. Control cultures show a cell death whose frequency     is nonsignificant     .sup.+  Student's "ttest.     NS = nonsignificant

                  TABLE 2    ______________________________________    CYTOTOXICITY OF THE CSF FROM PATIENTS SUFFERING    FROM MS OR OTHER NEUROLOGICAL DISEASE, DETECTED    USING THE BIOLOGICAL TEST ON A LINE OF    IMMORTALIZED ASTROCYTES AND QUANTIFIED BY    THE MTT COLORIMETRIC METHOD    PATIENT    No.                      CYTOTOXICITY    MS       MODE OF PROGRESSION                             (% dead cells)    ______________________________________    1        Remitting in exacerbation                             48.8 ± 4.5    2        Remitting in exacerbation                             54.6 ± 3.9    3        Remitting in exacerbation                             44.0 ± 6.5    4        Remitting in exacerbation                             39.8 ± 3.8    5        Remitting in exacerbation                             52.5 ± 2.9    6        Remitting in exacerbation                             68.5 ± 7.7    13       Chronic          7.2 ± 2.2    14       Chronic          8.2 ± 1.9    15       Chronic          4.1 ± 3.0    16       Chronic         0    17       Chronic          5.4 ± 1.7    18       Chronic          6.8 ± 0.8    NPH      (NON-MS CONTROLS)    19                       0    20                       0    21                       0    22                       0    23                       0    24                       0    ______________________________________     CSF: Cerebrospinal fluid; MTT: Methyltetrazolium; MS: Multiple sclerosis;     NPH: Normal pressure hydrocephalus. Cytotoxicity was measured with the     colorimetric technique using MTT after 96 h incubation. CSF samples were     diluted to 1:20 in the culture medium used for the immortalized     astrocytes. Each result represents the mean of two separate experiments     each representing a series of 5 independent wells, that is to say,     finally, a mean of 10 values per CSF tested. The CSF samples originated     from patients suffering from remitting form of MS one  sic! been drawn at     the time of clinical exacerbations. The difference in the mean     cytotoxicity values in the two subpopulations of MS, remitting versus     chronic, is statistically significant (p < 0.0001, MannWithney  sic! U     test), the difference between the results for MS CSF and that of controls     suffering from NPH is itself statistically significant (p < 0.0001,     MannWithney  sic! U test).

                  TABLE 3    ______________________________________    EXAMPLE OF PURIFICATION YIELD OF THE GLIOTOXIC    FACTOR FROM CULTURE SUPERNATANTS OF MONOCYTES    FROM MS PATIENTS    supernatant    volume (ml)               10.00    proteins (mg)             42.00    purification              1.0    cytotoxicity (mg)         1.2    yield (%)                100%    protein A-Sepharose    volume (ml)               8.00    proteins (mg)             35.30    purification              1.1    cytotoxicity (mg)         1.08    yield (%)                 93%    concanavalin A-Sepharose + NAP-25    volume (ml)               3.50    proteins (mg)             0.32    purification             100    cytotoxicity (mg)         0.012    yield (%)                 76%    ______________________________________

                                      TABLE 4    __________________________________________________________________________    DOSE-RESPONSE EFFECT: QUANTIFICATION    OF THE GLIOTOXIC ACTIVITY BY THE    Cr-51-RELEASE TEST                  BEFORE PURIFICATION                                 AFTER PURIFICATION                  % cytotoxicity % cytotoxicity    SAMPLE  DILUTION                  (in three measurements)                                 (in three measurements    __________________________________________________________________________    Culture medium            1/1   7.200                       10.000                            7.600                                 11.400                                      8.200                                           7.400    Control patient             1/20 11.400                       10.800                            10.200                                 7.100                                      6.600                                           8.200    MS 1     1/20 47.400                       42.800                            39.900                                 94.400                                      90.800                                           92.200    MS 1     1/200                  39.000                       40.400                            43.100                                 78.800                                      74.200                                           79.400    MS 1      1/1000                  36.200                       32.400                            33.300                                 72.100                                      70.400                                           70.000    MS 1      1/5000                  28.100                       26.600                            26.400                                 51.200                                      48.400                                           46.200    __________________________________________________________________________

                                      TABLE 5    __________________________________________________________________________    DOSE-RESPONSE EFFECT: QUANTIFICATION OF THE GLIOTOXIC    ACTIVITY BY THE MTT COLORIMETRIC TEST                  BEFORE PURIFICATION                                 AFTER PURIFICATION                  OD 570-630 nm  OD 570-630 nm    SAMPLE  DILUTION                  (in three measurements)                                 (in three measurements    __________________________________________________________________________    Culture medium            1/1   1.904                       1.870                            1.884                                 1.922                                      1.910                                           1.934    Control patient             1/20 1.912                       1.892                            1.897                                 1.882                                      1.864                                           1.890    MS 1     1/20 1.604                       1.572                            1.608                                 0.948                                      0.990                                           0.960    MS 1     1/200                  1.812                       1.794                            1.800                                 1.310                                      1.302                                           1.322    MS 1      1/1000                  1.890                       1.914                            1.888                                 1.760                                      1.784                                           1.762    MS 1      1/5000                  1.902                       1.896                            1.890                                 1.890                                      1.824                                           1.818    __________________________________________________________________________

BIBLIOGRAPHY

(1) Prineas J. W., The neuropathology of multiple sclerosis in "Handbookof Clinical Neurology: Demyelinating Diseases", volume 3 No. 47,Koetsier J. C. editor, 213-257, Elsevier, Amsterdam 1985.

(2) Prineas J. W., Barnard R. O., Kwon E. E., Sharer L. R. and Cho E.S., Multiple sclerosis: remyelination of nascent lesions. Ann. Neurol.,1993; 33, 137-151.

(3) Boyle E. A. and McGeer P. L., Cellular immune response in multiplesclerosis plaques, American Journal of Pathology, 1993; 137, 575-584.

(4) Charcot J. M., Histologie de la sclerose en plaques Histology ofmultiple sclerosis!, Gaz. Hop. (Paris), 1868; 41, 554-566.

(5) Hauw J. J. and Escourolle R., Aspects anatomopathologiques de lasclerose en plaques Anatomopathological aspects of multiple sclerosis!,in "La sclerose en plaques" Multiple sclerosis!, Rascol A., Bes A. andGuiraud-Chaumeil B. 9-47. Masson, Paris, 1980.

(6) Poirier J., Fleury J., and Gherardi R., La barrierehemato-encephalique, Donnees morphologiques The blood-brain barrier,Morphological data!, La Revue de Medecine Interne, 1983; 4, 131-144.

(7) Netsky M. G., and Shuangshoti S., The choroid plexus in health anddisease, University Press of Virginia, 1975.

(8) Gonzales-Scarano F., Grossman R. I., Galetta S. Atlas S. W. andSilberberg D. H., Multiple slerosis sic! disease activity correlateswith gadolinium enhancement magnetic resonance imaging, Ann. Neurol.,1987; 21, 300-306.

(9) Rapport S. I., Blood-brain barrier in physiology and medicine, RavenPress. 1976.

(10) Kent T. A. and McKendall R. R., Cerebral blood flow, cerebralmetabolism and blood-brain barrier, In, McKendall R. R. Ed., Handbook ofClinical Neurology, Vol. 12, No. 56: Viral disease, Elsevier, Amsterdam,1989.

(11) Prineas J. W. and Wright R. G., Macrophages, lymphocytes, andplasma cells in the perivascular compartment in chronic multiplesclerosis, Laboratory Investigation, 1978; 38, 409-421.

(12) Bergamini L, and Durell L., Multiple sclerosis, I, The immunepathogenetic hypothesis, Riv. Neurol., 1989; 59, 176-90.

(13) Calder V, Owen S, Watson C, Feldmann M, and Davidson A., MS: alocalized immune disease of central nervous system, Immunol Today, 1989;10, 99-103.

(14) Jervis G. A., and Koprowski H., Chronic experimental allergicencephalomyelitis, J. Neuropathol. Exp. Neurol., 1948; 7, 309-320.

(15) Prineas J. W., Pathology of early lesion in multiple sclerosis,Human pathology, 1975; 6, 23-7.

(16) Escourolle R., Hauw J. J. and Lyon-Caen O., Principales donneesmorphologiques, approches physiopathologiques et etiologiques de lasclerose en plaques Principal morphological data, physiopathological andetiological approaches to multiple sclerosis!, La Revue du Praticien(Paris), 1980; 30, 2047-2053.

(17) Mc Donald W. I., The mystery of the origin of multiple sclerosis,J. Neurol. Neurosurg. Psych., 1986; 49, 113-123.

(18) Carp R. I., Warner H. B. and Merz G. S., Viral etiology of multiplesclerosis., Prog. Med. Virol., 1978; 24, 158-177.

(19) Marie P., Sclerose en plaques et maladies infectieuses Multiplesclerosis and infectious diseases!, Le progres medical, 1884; 12,287-289.

(20) Gay D, Dick G, and Upson G., Multiple sclerosis caused by an oralspirochete?, Lancet; 1986, 2, 815-9.

(21) De Keyser J. Autoimmunity in multiple sclerosis. Neurology, 1988Mar, 38, 371-4.

(22) Juntunen J, Kinnunen E, Anti-Poika M, Koskenvuo M. Multiplesclerosis and occupational exposure to chemicals: a co-twin controlstudy of a nationwide series of twins, Br. J. Int. Med., 1989; 417-9.

(23) Ebers G. C., Bulman D., The geographic distribution of MS reflectsgenetic susceptibility, Neurology, 1986; 36, S1-108.

(24) Haegert D. G., Michaud M., Schwab C., Tansey C., Secary F., FrancisG., HLA-DR beta, -DQ alpha and -DQ beta restriction fragment lengthpolymorphisms in multiple sclerosis, J. Neurosci. Res., 1989; 23, 46-54.

(25) Waksman B. H., Mechanisms in Multiple Sclerosis, Nature, 1985; 318,104-105.

(26) Acha-Orbea H. and Palmer E., Mls--a retrovirus exploits the immunesystem, Immunology Today 1991; 12, 356-361.

(27) Cole B. C. and Atkin C. L., The mycoplasma arthritidis T-cellmitogen, MAM: a model superantigen, Immunology Today 1991; 12, 271-276.

(28) Rudge P., Does a retrovirally encoded superantigen cause multiplesclerosis?, Journal of Neurology, Neurosurgery and Psychiatry, 1991; 54,853-855.

(29) Woodland D. L., Happ M. P., Gollob K. J. and Palmer E., Anendogenous retrovirus mediating deletion of aβ T cells? Nature (London),1991; 349, 529-530.

(30) Traugott U., Multiple sclerosis: relevance of class I and class IIMHC-expressing cells to lesion development, Journal of Neuroimmunology,1987; 16, 283-302.

(31) Williams G. T. and Smith C. A., Molecular regulation of apoptosis:genetic controls on cell death, Cell, 1993; 74, 777-779.

(32) Levine B., Huang Q., Isaacs J. T., Reed J. C., Griffin D. E. andHardwick J. M., Conversion of lytic to persistent alphavirus infectionby the bcl-2 cellular oncogene, Nature, 1993; 361, 739-742.

(33) Newell M. K., VanderWall J., Beard K. S. and Freed J. H., Ligationof major histocompatibility complex class II molecules mediatesapoptotic cell death in resting B lymphocytes, P.N.A.S., 1993; 90,10459-10463.

(34) Selmaj. K. W. and Raine C. S., Tumor necrosis factor mediatesmyelin and oligodendrocyte damage in vitro, Ann. Neurol., 1988; 23,339-346.

(35) Barna B. P., Estes M. L., Jacobs B. S., Hudson S. and Ransohoff R.M., Human astrocytes proliferate in response to tumor necrosis factoralpha, J. Neuroimmunol., 1990; 30, 239-243

(36) Robbins D. S., Shirazi Y., Drysdale B. E., Lieberman A., Shin H. S.and Shin M. L., Production of cytotoxic factor for oligodendrocytes bystimulated astrocytes, The Journal of Immunology 1987; 139, 2593-2597.

(37) Beck J., Rondot P., Catinot L., Falcoff E., Kirchner J. andWietzerbin J., Increased production of interferon gamma and tumornecrosis factor precedes clinical manifestation in multiple sclerosis:do cytokine sic! trigger off exacerbations?, Acta Neurol. Scand., 1988;78, 318-323.

(38) Kaufmann S. H. E., Heat Shock Proteins and Immune Response, CurrentTopics in Microbiology and Immunology, vol. 167, Springer-Verlag,Berlin, 1991.

(39) Wienfield J. B., and Jarjour W. N., Stress proteins, autoimmunity,and autoimmune disease, in Heat Shock Proteins and Immune Response,Kaufmann S. H. E., Current Topics in Microbiology and Immunology, vol.167, 161-189, Springer-Verlag, Berlin, 1991.

(40) Brocke S., Gaur A., Piercy C., Gautam A., Gijbels K., Fathman C. G.and Steinman L., Induction of relapsing paralysis in experimentalautoimmune encephalomyelitis by bacterial superantigen, Nature, 1993;365, 642-644.

(41) Birnbaum G., Kotilinek L. and Albrecht L., Spinal fluid lymphocytesfrom a subgroup of multiple sclerosis patients respond to mycobacterialantigens, Ann. Neurol. 1993; 34, 18-24.

(42) Ransohoff R. M. and Rudick R. A., Heat-shock proteins andautoimmunity: implications for multiple sclerosis, Annals of Neurology,1993; 34, 5-7.

(43) Perron H. Geny C., Laurent A., et al., Leptomeningeal cell linefrom multiple sclerosis with reverse transcriptase activity and viralparticles, Res. Virol., 1989; 140, 551-561.

(44) Perron H., Geny C., Gratacap B., Laurent A., Mouriguand C., PellatJ., Perret J., and Seigneurin J. M., Isolation of an unknown retrovirusfrom CSF, blood and brain from patients with multiple sclerosis, in"Current concepts in multiple sclerosis", Wietholter et al., pp.111-116. Elsevier, Amsterdam, 1991.

(45) Perron H., Lalande B., Gratacap B., Laurent A., Genoulaz O., GenyC., Mallaret M., Schuller E., Stoebner P., and Seigneurin J. M.,Isolation of retrovirus from patients with multiple sclerosis, Lancet,1991; 337, 862-863.

(46) Dalgleish A. G., Fazakerley J. K. and Webb H. E., Do humanT-lymphotropic viruses (HTLVs) and other enveloped viruses induceautoimmunity in multiple sclerosis?, Neuropath. Appl. Neurobiol., 1987;13, 241-250.

(47) Birnbaum G., Kotilinek L. and Albrecht L., Spinal fluid lymphocytesfrom a subgroup of multiple sclerosis patients respond to mycobacterialantigens, Ann. Neurol., 1993; 34, 18-24.

(48) Davison A. N. and Sabri M. I., Biosynthesis of Myelin andNeurotoxic Factors in the Serum of Multiple Sclerosis Patients, Advancesin Experimental Medicine, vol. 100, 1978, New York, US, 19-25.

(49) Poser C. M. et al., New diagnostic criteria for multiple sclerosis:guidelines for research protocols, in "The diagnosis of multiplesclerosis", Poser C. M., Paty D. W., Scheinberg L., MacDonald W. I.,Ebers G. C. pp. 225-229, 1984, Thieme Stratton Inc., New York.

(50) Galiana E., Borde I., Marin P., Rassoulzadegan M., Cuzin F., GrosF., Rouget P. and Evrard C., Establishment of permanent astroglial celllines, able to differentiate in vitro, from transgenic mice carrying thepolyoma virus large-T gene: an alternative approach to brain cellimmortalization, Journal of Neuroscience Research, 1990; 26; 269-277.

(51) Mosmann T., Rapid calorimetric assay of cellular growth andsurvival: application to proliferation and cytotoxicity assays, J.Immunol. Meth., 1983; 65, 55-63.

(52) Wollinsky K. H., Hulser P. J., Mauch E., Mehrkens H. H. andKornhuber H. H., Liquorpherese bei 10 Patienten mit Multipler skleroseFluid pheresis in 10 patients with multiple sclerosis! in verhandlungensic! der Deutschen Gesellschaft fur Neurologie, Grundmann M et al., vol7 (1992) Saarbrucken.

(53) Bradford M. M., A rapid sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle of protein-dyebinding, Anal. Biochem., 1976; 72, 248-254.

(54) Silberberg D. et al., Tissue culture demyelination by normal humanserum, Annals of Neurology, 1984: 15, 575-580.

What is claimed is:
 1. An isolated or purified protein gliotoxic factorhaving toxic activity with respect to astrocytic cells, wherein saidtoxic activity is expressed as cell death by apoptosis.
 2. The gliotoxicfactor according to claim 1, wherein said toxic activity is associatedwith at least one globular glycoprotein.
 3. The gliotoxic factoraccording to claim 1, comprising a light fraction centered around anapparent molecular weight of approximately 17 kD, and a less abundantheavy fraction centered around an apparent molecular weight ofapproximately 21 kD, at least said light fraction being resistant, undernondenaturing conditions, to a hydrolytic action of at least one enzymeselected from the group consisting of pronase, trypsin and proteinase K,each of the two said fractions displaying affinity for at least onelectin.
 4. A method for obtaining an isolated or purified gliotoxicfactor having toxic activity with respect to astrocytic cells, whereinsaid toxic activity is expressed as cell death, said methodcomprising:obtaining a biological sample, treating said sample on an ionexchange resin, and treating said ion-exchanged sample on a column forseparation by exclusion, to obtain said gliotoxic factor.
 5. A gliotoxicfactor according to claim 3, wherein said at least one lectin isconcanavalin A.
 6. The method according to claim 4, wherein said sampleis obtained from a patient suffering from an autoimmune disorder.
 7. Anisolated or purified gliotoxic factor having an apparent molecularweight of approximately 17 kD, that is resistant, under nondenaturingconditions, to a hydrolytic action of at least one enzyme selected fromthe group consisting of pronase, trypsin and proteinase K, and thatdisplays affinity for at least one lectin, said gliotoxic factor havingtoxic activity with respect to astrocytic cells, wherein said toxicactivity is expressed as cell death.
 8. The gliotoxic factor accordingto claim 1, having an apparent molecular weight of approximately 21 kDand displaying affinity for at least one lectin.
 9. An isolated orsynthetic antibody specific to the gliotoxic factor according toclaim
 1. 10. The method according to claim 4, wherein said sample isobtained from a patient suffering from a neurological disorder.
 11. Themethod according to claim 4, wherein said sample is obtained from apatient suffering from multiple sclerosis.
 12. The method according toclaim 11, wherein said sample is obtained from serum or spinal fluid ofsaid patient.
 13. The gliotoxic factor according to claim 1, whereinsaid toxic activity is furthermore expressed as at least one ofcytomorphological disorganization of a network of intermediate filamentsof said astrocytic cells and protein degradation of said network ofintermediate filaments.
 14. The method according to claim 4, whereinsaid gliotoxic factor comprises a light fraction centered around anapparent molecular weight of approximately 17 kD, and a less abundantheavy fraction centered around an apparent molecular weight ofapproximately 21 kD, at least said light fraction being resistant, undernondenaturing conditions, to a hydrolytic action of at least one enzymeselected from the group consisting of pronase, trypsin and proteinase K,each of the two said fractions displaying affinity for at least onelectin.
 15. The method according to claim 4, wherein said cell death isby apoptosis.
 16. The gliotoxic factor according to claim 7, whereinsaid cell death is by apoptosis.
 17. A pharmaceutical or diagnosticcomposition comprising a pharmaceutical- or diagnostic-effective amountof a protein gliotoxic factor having toxic activity with respect toastrocytic cells, wherein the toxic activity is expressed as cell deathby apoptosis.
 18. A pharmaceutical or diagnostic composition comprisinga ligand specific to a protein gliotoxic factor having toxic activitywith respect to astrocytic cells, wherein the toxic activity isexpressed as cell death by apoptosis.
 19. The method according to claim14, wherein said at least one lectin is concanavalin A.
 20. Thepharmaceutical or diagnostic composition according to claim 18, whereinsaid ligand is an antibody.